Phenylahistin and the phenylahistin analogs, a new class of anti-tumor compounds

ABSTRACT

Methods of using a compound, its pharmaceutically acceptable salts, and/or its pro-drug esters, in isolated form, to treat cancer, and methods for isolating, for formulating, and for administering the compound, salt, and/or pro-drug ester as an antitumor agent, wherein the compound, salt, or pro-drug ester has the following structure:  
                 
 
     wherein:  
     R 1 , R 2 , R 5 , R 7 , and R 8  are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C 1 -C 24  alkyl, unsaturated C 1 -C 24  alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, substituted nitro, phenyl, and substituted phenyl groups,  
     R 3 , R 4 , and R 6  are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C 1 -C 12  alkyl, unsaturated C 1 -C 12  alkenyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups,  
     X 1  and X 2  are separately selected from the group consisting of an oxygen atom, and a sulfur atom, and  
     the dashed bond represents a bond selected from the group consisting of a carbon-carbon single bond and a carbon-carbon double bond. Most preferably, R 3  and R 4  are hydrogen, and each are involved in hydrogen bonds, and/or the dashed bond is a double bond, such that the chemical backbone of the compound substantially retains a substantially planar conformation.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/995,851, filed Nov. 27, 2001, which is a continuation of,and claims priority from, U.S. application Ser. No. 09/440,316, filedNov. 12, 1999, U.S. Pat. No. 6,358,957, which application claimedpriority from U.S. Provisional Application Ser. No. 60/108,211,PHENYLAHISTIN AS AN ANTI-TUMOR COMPOUND, filed Nov. 12, 1998, byFukumoto et al., and also claimed priority from U.S. ProvisionalApplication Ser. No. 60/108,736, PHENYLAHISTIN AS AN ANTI-TUMORCOMPOUND, filed Nov. 17, 1998, by Fukumoto et al., Each of theabove-mentioned applications is incorporated herein by reference in itsentirety.

[0002] This application is also related to U.S. Application Ser. No.__/___,___, entitled DEHYDROPHENYLAHISTINS AND ANALOGS THEREOF AND THESYNTHESIS OF DEHYDROPHENYLAHISTINS AND ANALOGS THEREOF, filed on thisdate, and which is incorporated hereby by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to the fields of chemistry andmedicine. More specifically, the present invention relates to compoundsand procedures useful in the treatment of cancer, or in the treatment offungal infections.

[0005] 2. Description of the Related Art

[0006] It is thought that a single, universal cellular mechanismcontrols the regulation of the eukaryotic cell cycle process. See, e.g.,Hartwell, L. H. et al., Science (1989), 246: 629-34. It is also knownthat when an abnormality arises in the control mechanism of the cellcycle, cancer or an immune disorder may occur. Accordingly, as is alsoknown, antitumor agents and immune suppressors may be among thesubstances that regular the cell cycle. Thus, new eukaryotic cell cycleinhibitors are needed as antitumor and immune-enhancing compounds, andshould be useful in the treatment of human cancer as chemotherapeutic,anti-tumor agents. See, e.g., Roberge, M. et al., Cancer Res. (1994),54, 6115-21.

[0007] Recently, it has been reported that tryprostatins A and B (whichare diketopiperazines consisting of proline and isoprenylated tryptophanresidues), and five other structurally-related diketopiperazines,inhibited cell cycle progression in the M phase, see Cui, C. et al., J.Antibiotics (1996), 49, 527-33; Cui, C. et al. J. Antibiotics (1996),49, 534-40, and that these compounds also affect the microtubuleassembly, see Usui, T. et al. Biochem J. (1998) 333, 543-48; Kondon, M.et al. J. Antibiotics (1998) 51, 801-04. Furthermore, natural andsynthetic compounds have been reported to inhibit mitosis, thus inhibitthe eukaryotic cell cycle, by binding to the colchicine binding-site(CLC-site) on tubulin, which is a macromolecule that consists of two 50kDa subunits (α- and β-tubulin) and is the major constituent ofmicrotubules. See, e.g., Iwasaki, S., Med. Res. Rev. (1993) 13, 183-198;Hamel, E. Med. Res. Rev. (1996) 16, 207-31; Weisenberg, R. C. et al.,Biochemistry (1969) 7, 4466-79. Microtubules are thought to be involvedin several essential cell functions, such as axonal transport, cellmotility and determination of cell morphology. Therefore, inhibitors ofmicrotubule function may have broad biological activity, and beapplicable to medicinal and agrochemical purposes. It is also possiblethat colchicine (CLC)-site ligands such as CLC, steganacin, see Kupchan,S. M. et al., J. Am. Chem. Soc. (1973) 95, 1335-36, podophyllotoxin, seeSackett, D. L., Pharmacol. Ther. (1993) 59, 163-228, andcombretastatins, see Pettit, G. R. et al., J. Med. Chem. (1995) 38,166-67, may prove to be valuable as eukaryotic cell cycle inhibitorsand, thus, may be useful as chemotherapeutic agents.

[0008] Although diketopiperazine-type metabolites have been isolatedfrom various fungi as mycotoxins, see Horak R. M. et al., J.C.S. Chem.Comm. (1981) 1265-67; Ali M. et al., Toxicology Letters, (1989) 48,235-41, or as secondary metabolites, see Smedsgaard J. et al., J.Microbiol. Meth. (1996) 25, 5-17, little is known about the specificstructure of the diketopiperazine-type metabolites and their antitumoractivity, particularly in vivo. Furthermore, even though known antitumorsubstances isolated from microorganism metabolites (includinganthracyclins and mitomycins that exhibit antitumor activity by bindingto DNA) have been used as antitumor agents, see MicroorganicPharmaceutical Chemistry, revised 2nd edition, edited by Yoshio Ueno &Satoshi Ohmura, Nankohdo Publishing Co., (1986)), and even thoughanti-tumor substances having non-DNA binding operating mechanism havebeen isolated from microorganism metabolites, see Minoru Yoshida, M.Protein Nucleic Acid Enzymes (1993) 38, 1753; and Iwasaki, N., Chemistryand Living Organisms, (1994) 32, No. 3, 153, there is a particular needfor new microorganism metabolite-derived compounds having animalcell-specific proliferation-inhibiting activity and high antitumoractivity and selectivity. There is therefore a related need forsubstantially purified, and structurally and biologically characterizedfungal diketopiperazine-type metabolites and fungaldiketopiperazine-type metabolite-derivatives.

SUMMARY OF THE INVENTION

[0009] A compound, and any pharmaceutically acceptable salt or pro-drugester thereof, suitable for use as an anti-tumor agent having thefollowing generic structure:

[0010] wherein:

[0011] R₁, R₂, R₅, R₇, and R₈ are each separately selected from thegroup consisting of a hydrogen atom, a halogen atom, and saturatedC₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, amino, substituted amino, nitro, substituted nitro, phenyl,and substituted phenyl groups, and

[0012] R₃, R₄, and R₆ are each separately selected from the groupconsisting of a hydrogen atom, a halogen atom, and saturated C₁-C₁₂alkyl, unsaturated C₁-C₁₂ alkenyl, cycloalkyl, alkoxy, cycloalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino,substituted amino, nitro, and substituted nitro groups,

[0013] X₁ and X₂ are separately selected from the group consisting of anoxygen atom, and a sulfur atom, and

[0014] the dashed bond represents a bond selected from the groupconsisting of a carbon-carbon single bond and a carbon-carbon doublebond.

[0015] In a preferred embodiment, the invention comprises, insubstantially purified form, the compound herein named alternatively“(−)-phenylahistin,” “(−)-NSCL-96F037,” or “(−)-PLH,” which is adiketopiperazine composed of L-phenylalanine and isoprenylateddehydrohistidine. This compound has the following stereo-specificchemical structure:

[0016] The invention also comprises a method of treating cancercomprising administering an effective tumor-growth-inhibiting amount ofthe compound, and any pharmaceutically acceptable salt or pro-drug esterof generic structure (I), and preferably, phenylahistin, and morepreferably, (−)-phenylahistin, and pharmaceutically acceptable salt andpro-drug esters thereof. In preferred embodiments of the compound, salt,or pro-drug ester of the present invention, R₃ and R₄ are hydrogen, andeach are involved in hydrogen bonds, and/or the dashed bond is a doublebond.

[0017] Other embodiments relate to methods of treating and/or preventingat least one fungal infection in a mammal afflicted with at least onefungal infection. The methods can include, for example, administering anantifungally effective amount of a compound sufficient for such treatingor preventing. The compound can have, for example, the followingstructure:

[0018] wherein:

[0019] R₁, R₂, R₅, R₇, and R₈ are each separately selected from thegroup consisting of a hydrogen atom, a halogen atom, and saturatedC₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, amino, substituted amino, nitro, substituted nitro, phenyl,and substituted phenyl groups,

[0020] R₃, R₄, and R₆ are each separately selected from the groupconsisting of a hydrogen atom, a halogen atom, and saturated C₁-C₁₂alkyl, unsaturated C₁-C₁₂ alkenyl, cycloalkyl, alkoxy, cycloalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino,substituted amino, nitro, and substituted nitro groups,

[0021] X₁ and X₂ are separately selected from the group consisting of anoxygen atom, and a sulfur atom, and

[0022] the dashed bond represents a bond selected from the groupconsisting of a carbon-carbon single bond and a carbon-carbon doublebond.

[0023] The fungal infection can be for example, an Aspergillosisinfection, a blastomycosis infection, a Candidiasis infection, aCoccidioidomycosis infection, a Cryptococcosis infection, aHistopolasmosis infection, a Paracoccidioidomycosis, aSporotrichosisand, a Mucormycosis infection, and the like. Preferably,the Aspergillosis infection can be invasive pulmonary aspergillosis.Further, preferably, the Mucormycosis infection can be craniofacialmucormycosis, pulmonary mucormycosis and the like. Also, preferably, theCandidiasis infection can be retrograde candidiasis of the urinarytract, for example.

[0024] Still further embodiments relate to pharmaceutical compositionsfor treating or preventing fungal infection. The compositions caninclude, for example, an antifungally effective amount of apharmaceutically acceptable carrier and a compound having the structure:

[0025] wherein:

[0026] R₁, R₂, R₅, R₇, and R₈ are each separately selected from thegroup consisting of a hydrogen atom, a halogen atom, and saturatedC₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, amino, substituted amino, nitro, substituted nitro, phenyl,and substituted phenyl groups,

[0027] R₃, R₄, and R₆ are each separately selected from the groupconsisting of a hydrogen atom, a halogen atom, and saturated C₁-C₁₂alkyl, unsaturated C₁-C₁₂ alkenyl, cycloalkyl, alkoxy, cycloalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino,substituted amino, nitro, and substituted nitro groups,

[0028] X₁ and X₂ are separately selected from the group consisting of anoxygen atom, and a sulfur atom, and

[0029] the dashed bond represents a bond selected from the groupconsisting of a carbon-carbon single bond and a carbon-carbon doublebond.

[0030] Preferably, the fungal infection can be an Aspergillosisinfection, a blastomycosis infection, a Candidiasis infection, aCoccidioidomycosis infection, a Cryptococcosis infection, aHistopolasmosis infection, a Paracoccidioidomycosis, a Sporotrichosisand, a Mucormycosis infection, and the like. Preferably, theAspergillosis infection can be invasive pulmonary aspergillosis.Further, preferably, the Mucormycosis infection can be craniofacialmucormycosis, pulmonary mucormycosis and the like. Also, preferably, theCandidiasis infection can be retrograde candidiasis of the urinarytract, for example.

[0031] Embodiments also relate to methods and materials for treating orpreventing cancers and infection by a pathogenic fungus in a mammaliansubject, particularly a human patient, by administering to the subject aphenylahistin or its analog. The methods can include administering tothe subject a composition comprising an effective antitumor orantifungal amount of a phenylahistin or its analog.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which are incorporated in and formpart of the specification, merely illustrate embodiments of the presentinvention. Together with the remainder of the specification, they aremeant to serve to explain preferred modes of making certain compounds ofthe invention to those of skilled in the art. In the drawings:

[0033]FIG. 1 illustrates Reaction Scheme 1, which shows the preparationof certain reduced derivatives of (−)- and (+)-phenylahistin from theenantiomers of phenylahistin.

[0034]FIG. 2 illustrates Reaction Scheme 2, which shows a syntheticmethod for producing a compound of the invention, PLH-Cl, fromH₂N-Phe-Gly-OMe. (The asterisk indicates that racemation occurred;(−):(+)=79:21.).

[0035]FIG. 3 illustrates Reaction Scheme 3, which shows the preparationof two reduced derivatives of (−)-phenylahistin, compounds identified as(−)-12 and (−)-13, which are respectively, identical to compounds 3 and5 of FIG. 1. In FIG. 3, (a) refers to H₂/10% Pd—C, MeOH, roomtemperature, reaction time of 2 hours, and (b) refers to H₂/10% Pd—C,MeOH, room temperature, reaction time of 24 hours.

[0036]FIG. 4 illustrates Reaction Scheme 4, which shows the preparationof two N-methylated derivatives of (−)-phenylahistin. In FIG. 4, (a)refers to MeI, NaH, DMF, −30° C., reaction time of 2 hours, (b) refersto MeI, NaH, DMF, room temperature, reaction time of 2 hours; and (c)refers to chiral HPLC separation as described herein.

[0037]FIG. 5 illustrates Reaction Scheme 5, which shows the preparationof two derivatives of (−)-phenylahistin, compounds identified as 19 and20, which is the (−)-enantiomer of compound 9 of FIG. 2. In FIG. 5, (a)refers to MeOH at reflux for 14 hours, (b) refers to AcO₂, AcONa, 80°C., reaction time of 14 hours, and (c-e) refers to reaction in a mixtureof compound 18, LDA, HMPA, DMF, at −60° C. for 30 minutes, followed bycooling to room temperature and placing in a solution of Tf₂O, pyridinefor 10 minutes, followed by overnight reaction in NH₄OH at roomtemperature.

[0038]FIG. 6 depicts the effect of t-butyl-phenylahistin in comparisonto colchicine and taxol on HuVEC monolayer permeability to FITC-Dextran.

[0039]FIG. 7 depicts the effects of t-butyl-phenylahistin alone and incombination with CPT-11 on estimated tumor growth in the HT-29 humancolon tumor xenograft model.

[0040]FIG. 8 depicts the effects of t-butyl-phenylahistin alone and incombination with CPT-11 on the weight of tumors excised at autopsy inindividual mice in the HT-29 Human Colon Tumor Xenograft model.

[0041]FIG. 9 depicts the effects of t-butyl-phenylahistin alone and incombination with Taxotere on the estimated tumor growth based onobservations made during the in-life portion of the DU-145 HumanProstate Tumor Xenograft Model.

[0042]FIG. 10 depicts the effects of t-butyl-phenylahistin alone and incombination with Taxotere in MCF-7 Human Breast Tumor Xenograft model.

[0043]FIG. 11 depicts effects of t-butyl-phenylahistin alone and incombination with Paclitaxel in the Murine Melanoma B16 F10 MetastaticTumor Model.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0044] Numerous references are cited herein. These references are to beconsidered incorporated by reference into this specification.

[0045] Objects of this invention include (1) providing a class of newcompounds, including pharmaceutically acceptable salts of the class ofcompounds, that exhibit animal cell-specific proliferation inhibiting,tumor-growth inhibiting activity, anti-fungal activity, and/orcell-cycle inhibiting activity, and (2) providing a class of fungi forproducing, and (3) providing a method for producing said class ofcompounds, as well as a class of pharmaceutically acceptable cell cycleinhibitors, antitumor agents, and anti-fungal agents comprising saidcompounds and/or their derivatives as active ingredients. It is also anobject of this invention to provide a method of treating cancer,particularly human cancer, comprising the step of administering aneffective tumor-growth inhibiting amount of a member of a class of newanti-tumor compounds. Further, it is an object of this invention toprovide a method of treating fungal infections or pathogenic fungus,including those affecting animals and humans, comprising the step ofadministering an effective tumor-growth inhibiting amount of a member ofa class of new anti-tumor compounds. In the preferred embodiment of thepresent invention, but not necessarily in all embodiments of the presentinvention, these objectives are simultaneously met.

[0046] The invention provides the compound represented by Formula (I):

[0047] wherein:

[0048] R₁, R₂, R₅, R₇, and R₈ are each separately selected from thegroup consisting of a hydrogen atom, a halogen atom, saturated C₁-C₂₄alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,amino, substituted amino, nitro, substituted nitro, phenyl, andsubstituted phenyl groups, all having, where applicable, up to 24 carbonatoms,

[0049] R₃, R₄, and R₆ are each separately selected from the groupconsisting of a hydrogen atom, a halogen atom, and saturated C₁-C₁₂alkyl, unsaturated C₁-C₁₂ alkenyl, cycloalkyl, alkoxy, cycloalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino,substituted amino, nitro, and substituted nitro groups, all having,where applicable, up to 12 carbon atoms,

[0050] X₁ and X₂ are separately selected from the group consisting of anoxygen atom, and a sulfur atom, and the dashed bond represents either acarbon-carbon single bond or a carbon-carbon double bond, of anytertiary conformation, in a particular embodiment of the invention.

[0051] The invention also provides pharmaceutically acceptable salts andpro-drug esters of the compound of Formula (I).

[0052] The term “pro-drug ester,” especially when referring to apro-drug ester of the compound of Formula (I), refers to a chemicalderivative of the compound that is rapidly transformed in vivo to yieldthe compound, for example, by hydrolysis in blood. The term “pro-drugester” refers to derivatives of the compound of the present inventionformed by the addition of any of several ester-forming groups that arehydrolyzed under physiological conditions. Examples of pro-drug estergroups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl andmethoxymethyl, as well as other such groups known in the art, includinga (5-R-2-oxo-1,3-dioxolen-4-yl)methyl group. Other examples of pro-drugester groups can be found in, for example, T. Higuchi and V. Stella, in“Pro-drugs as Novel Delivery Systems”, Vol. 14, A.C.S. Symposium Series,American Chemical Society (1975); and “Bioreversible Carriers in DrugDesign: Theory and Application”, edited by E. B. Roche, Pergamon Press:New York, 14-21 (1987) (providing examples of esters useful as prodrugsfor compounds containing carboxyl groups).

[0053] The term “pharmaceutically acceptable salt,” especially whenreferring to a pharmaceutically acceptable salt of the compound ofFormula (I), refers to any pharmaceutically acceptable salts of acompound, and preferably refers to an acid addition salt of a compound.Preferred examples of pharmaceutically acceptable salt are the alkalimetal salts (sodium or potassium), the alkaline earth metal salts(calcium or magnesium), or ammonium salts derived from ammonia or frompharmaceutically acceptable organic amines, for example C₁-C₇alkylamine, cyclohexylamine, triethanolamine, ethylenediamine ortris-(hydroxymethyl)-aminomethane. With respect to compounds of theinvention that are basic amines, the preferred examples ofpharmaceutically acceptable salts are acid addition salts ofpharmaceutically acceptable inorganic or organic acids, for example,hydrohalic, sulfuric, phosphoric acid or aliphatic or aromaticcarboxylic or sulfonic acid, for example acetic, succinic, lactic,malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic,p-toluensulfonic or naphthalenesulfonic acid.

[0054] Preferred pharmaceutical compositions of the present inventioninclude pharmaceutically acceptable salts and pro-drug esters of thecompound of Formula (I). Accordingly, if the manufacture ofpharmaceutical formulations involves intimate mixing of thepharmaceutical excipients and the active ingredient in its salt form,then it is preferred to use pharmaceutical excipients which arenon-basic, that is, either acidic or neutral excipients.

[0055] In preferred embodiments of the compounds of the presentinvention, a relatively rigid, planar pseudo three-ring structure isformed. To stable such a relatively rigid, planar pseudo three-ringstructure, R₃ and R₄ are hydrogen, each involved in hydrogen bonds.Furthermore, such a relatively rigid, planar pseudo three-ring structureis stabilized wherein the dashed bond is a double bond.

[0056] The term “halogen atom” means any one of the radio-stable atomsof column 17 of the Periodic Table of the Elements, i.e., fluorine,chlorine, bromine, or iodine, with fluorine and chlorine beingpreferred.

[0057] The term “alkyl” means any unbranched or branched, substituted orunsubstituted, saturated hydrocarbon, with C₁-C₆ unbranched, saturated,unsubstituted hydrocarbons being preferred, with methyl, ethyl,iosbutyl, and tert-butyl being most preferred. Among the substituted,saturated hydrocarbons, C₁-C₆ mono- and di- and per-halogen substitutedsaturated hydrocarbons and amino-substituted hydrocarbons are preferred,with perfluromethyl, perchloromethyl, perfluoro-tert-butyl, andperchloro-tert-butyl being the most preferred. The term “cycloalkyl”refers to any non-aromatic hydrocarbon ring, preferably having five totwelve atoms comprising the ring.

[0058] The term “alkenyl” means any unbranched or branched, substitutedor unsubstituted, unsaturated hydrocarbon including polyunsaturatedhydrocarbons, with C₁-C₆ unbranched, mono-unsaturated anddi-unsaturated, unsubstituted hydrocarbons being preferred, andmono-unsaturated, di-halogen substituted hydrocarbons being mostpreferred. In the R₁ and R₈ positions, of the compound of structure (I)a z-isoprenyl moiety is particularly preferred. The term “cycloalkenyl”refers to any non-aromatic hydrocarbon ring, preferably having five totwelve atoms comprising the ring.

[0059] The terms “aryl,” “substituted aryl,” “heteroaryl,” and“substituted heteroaryl” refer to aromatic hydrocarbon rings, preferablyhaving five, six, or seven atoms, and most preferably having six atomscomprising the ring. “Heteroaryl” and “substituted heteroaryl,” refer toaromatic hydrocarbon rings in which at least one heteroatom, e.g.,oxygen, sulfur, or nitrogen atom, is in the ring along with at least onecarbon atom.

[0060] The term “alkoxy” refers to any unbranched, or branched,substituted or unsubstituted, saturated or unsaturated ether, with C₁-C₆unbranched, saturated, unsubstituted ethers being preferred, withdimethyl, diethyl, methyl-isobutyl, and methyl-tert-butyl ethers beingmost preferred. The term “cycloalkoxy” refers to any non-aromatichydrocarbon ring, preferably having five to twelve atoms comprising thering.

[0061] The terms “purified,” “substantially purified,” and “isolated” asused herein refer to the compound of the invention being free of other,dissimilar compounds with which the compound of the invention isnormally associated in its natural state, so that the compound of theinvention comprises at least 0.5%, 1%, 5%, 10%, or 20%, and mostpreferably at least 50% or 75% of the mass, by weight, of a givensample. In one preferred embodiment, these terms refer to the compoundof the invention comprising at least 95% of the mass, by weight, of agiven sample.

[0062] The terms “anti-tumor” and “tumor-growth-inhibiting,” whenmodifying the term “compound,” and the terms “inhibiting” and“reducing”, when modifying the terms “compound” and/or the term “tumor,”mean that the presence of the subject compound is correlated with atleast the slowing of the rate of growth of the tumor. More preferably,the terms “anti-tumor,” “tumor-growth-inhibiting,” “inhibiting,” and“reducing” refer to a correlation between the presence of the subjectcompound and at least the temporary cessation of tumor growth. The terms“anti-tumor,” “tumor-growth-inhibiting,” “inhibiting,” and “reducing”also refer to, particularly in the most preferred embodiment of theinvention, a correlation between the presence of the subject compoundand at least the temporary reduction in the mass of the tumor.

[0063] The invention also provides fungi of the genus Aspergillus thatare capable of producing the anti-tumor compound of the invention, andspecifically, fungi that are capable of producing the anti-tumorcompound of the invention wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ areeach a hydrogen atom, and X₁ and X₂ are each separately an oxygen atom.Preferably, the fungi is an Aspergillus ustus capable of producing theantitumor compound of the invention in an amount of not less than 2 mg,preferably not less than 8 mg, per liter of the production medium asmeasured, by high performance liquid chromatography using the compoundrepresented by Formula (I) as a standard with respect to what isobtained by placing, in 18 mm-diameter, 180 mm-length test tubes, 5 mlof production medium (production medium: glucose 5 g/l, glycerin 20ml/l, cotton seed lees 20 g/l, yeast extract 2 g/l, sodium chloride 2.5g/l, calcium carbonate 4 g/l (pH6.5)), and then inoculating each testtube with 50 μl of a fungus suspension prepared by suspending conidia ofthe fungal strain in sterilized water, incubating the culture broth for5 days at 27° C. under reciprocal shaking (260 rpm), adding 10 ml ofacetone to the culture broth in each test tube, effecting extraction for1 day at room temperature, removing impurities by filtration,vacuum-concentrating the filtrate to distill off the acetone, makingthree additions of 5 ml of ethyl acetate to the remaining water layer toeffect extraction, effecting vacuum-concentration and drying to obtain asolid, and dissolving the solid in methanol. The invention also providesa method of producing the anti-tumor compound of Formula (I) comprisingthe steps of incubating in a culture medium a microorganism belonging tothe genus Aspergillus that is capable of producing the antitumorcompound represented of Formula (I) and collecting the compound from theculture. The invention further provides a cell cycle inhibitor and anantitumor agent containing the antitumor substance of Formula (I) as anactive ingredient, in combination with a pharmaceutically acceptablecarrier or excipient.

[0064] Most specifically, a preferred embodiment of the inventionprovides, phenylahistin, which exhibits the following physical andchemical characteristics:

[0065] (i) molecular weight: 350 (FABMS M/Z 351 (M+H)),

[0066] (ii) molecular formula: C₂₀H₂₂N₄O₂,

[0067] (iii) infrared absorption spectrum (IR V_(max) (KBr)(cm⁻¹)):3440, 3240, 1670, 1640, 1140;

[0068] (iv) ¹H-nuclear magnetic resonance spectrum (500 MHz, measured inCDCl₃, chemical shift value of CHCl₃ set to 7.24 ppm as internalstandard) having intensities at (δ (ppm)): 1.47(6H, s), 2.94 (1H, dd,J=14, 10 Hz), 3.45(1H, dd, J=14, 4 Hz), 4.33(1H, brd, J=10 Hz), 5.10(1H,d, J=17 Hz), 5.14(1H, d, J−11 Hz), 5.88(1H, br), 6.00(1H, dd, J=17, 11Hz), 6.86(1H, s), 7.21-7.26(3H, m), 7.31(2H, t, J=8 Hz), 7.53(1H, s),9.61(1H, br), 12.08(1H,br);

[0069] (v) ¹³C-nuclear magnetic resonance spectrum (400 MHz, measured inCDCl₃, chemical shift value of CDCl₃ set to 77.10 ppm as internalstandard) having intensities at (δ (ppm)): 28.07(CH₃), 28.07(CH₃),37.69(C), 41.33(CH₂), 57.24(CH), 105.67(CH), 113.46(CH₂), 123.77(CH),127.56(CH), 129.18(CH), 129.18(CH), 129.62(CH), 129.62(CH), 132.29(C),132.64(C), 135.55(C), 136.88(C), 144.75(CH), 160.04(C), 164.85(C);

[0070] (vi) ¹⁵N-nuclear magnetic resonance spectrum (600 MHz, measuredin CDCl₃, chemical shift value of ammonia set to 0 ppm as internalstandard) having intensities at (δ (ppm)): 112, 134, 161, 253;

[0071] (vii) maximum absorption value of ultraviolet spectrum inmethanol at 230 nm, and under neutral condition at 323 nm;

[0072] (viii) excited under a neutral condition in methanol by 320-340nm ultraviolet light, having a maximum value at 395-400 nm and emittingfluorescence having 350-550 nm wavelength width;

[0073] (ix) soluble in ethyl acetate, chloroform, methanol, andpyridine, but only slightly soluble in water, benzene, and toluene;

[0074] (x) negative in ninhydrin reaction, positive in color reactionwith nitrous acid (orange); and

[0075] (xi) substance color: white.

[0076] According to the examples provided herein, phenylahistin has beenisolated from Aspergillus ustus NSC-F038 and NSC-F037, two new strainsof fungus; this phenylahistin exhibits cytotoxic and cell cycleinhibitory activities. Both of these strains of fungi have beendeposited with the National Institute of Bioscience andHuman-Technology, Japan, Agency of Industrial Science and Technology,Ministry of International Trade and Industry. These strains exhibit thefollowing mycological characteristics:

[0077] (1) Growth morphology in various culture media: Growth onCzapek's yeast extract agar medium (25° C.) is rapid, reaching 45-46 mmin 7 days. Growth on Czapek's yeast extract agar medium (37° C.) issomewhat slower, reaching 39-41 mm in 7 days. On malt extract agarmedium, colony surface is gray and colony rear surface grayish-green.

[0078] (2) Morphological properties.

[0079] Morphological properties on Czapek's agar medium are indicated.

[0080] Condial heads: Radiate

[0081] Conidiophore: Smooth surface, brown with length of 100-350:m,4-7:m diameter

[0082] Vesicles: Spherical-flask-shaped, 11-15:m diameter, upper ⅔-½forms metulae

[0083] Metulae: covering upper half to two-thirds of the vesicles,5-7×4-7 μm

[0084] Phialides: amplliform, 5-8×3-4:m

[0085] Conidium: Brown, sperical, rough wall, 3×5:m

[0086] From these mycological properties, Aspergillus ustus NSC-F037 andAspergillus ustus NSC-F038 were found, in accordance with the textAspergillus, K. B. Raper and D. I. Fennel, Williams and Wilkins (1965),to belong to subphylum Fungi Imperfecti, order monilliales, genusAspergillus, species ustus. The strains were therefore termedAspergillus ustus NSC-F037 and Aspergillus ustus NSC-F038. It wasconcluded that Aspergillus ustus NSC-037 and Aspergillus ustus NSC-038were both novel fungal strains, and they were respectively assigned theacquisition numbers FERM P-15829 and FERM P-15930.

[0087] The compound of Formula (I), the compound of the invention, canbe produced by ordinary methods, from a culture medium and viaextracting the compound from a culture of either Aspergillus ustusNSC-037 or Aspergillus ustus NSC-038 in the following manner. Althoughthe culture can be either liquid or solid, industrially advantageousculture can be achieved by inoculating a liquid medium with a fungussuspension of the microorganism and incubating the medium underaeration-mixing. Although the culture medium nutrients are notparticularly limited, carbon sources, nitrogen sources and other culturemedium ingredients ordinarily used to culture microorganisms may beincluded and are preferred. Usable carbon sources include starch,glycerin, glucose, sucrose, galactose, and the like. Usable nitrogensources included peptone, soy bean powder, meat extract, corn-steepedliquor, cotton seed lees, ammonium salt, nitrates, and other organic andinorganic nitrogen compounds. In addition, inorganic salts and tracenutrients may be added as will be deemed appropriate by those of skillin the art. The culture temperature, culture time period, and otherculture conditions are preferably chosen as conditions that areappropriate for growth of the selected fungus, and also to maximizeproduction of the compound represented by Formula (1). For instance, thepH of the culture medium is preferably between approximately 4 andapproximately 9, and more preferably between 5 and 8, and the culturetemperature is preferably between 15 and 35° C., and more preferablybetween 23 and 28° C. The culture period is preferably between 48 and192 hours, and more preferably between 72 and 192 hours. However, theculture medium composition, culture medium pH, culture mediumtemperature, culture period and. other culture conditions shouldnaturally be appropriately adjusted according to the type of fungusused, the external conditions, and the like so as to obtain the desiredresults of producing the compound of the present invention. The compoundrepresented by Formula (1) can be collected from such a culture byappropriate use of means ordinarily used for collecting metabolites. Forexample, any means utilizing the difference in affinity for and organicsolvent between the compound represented by Formula (1) and othersubstances contained in the culture, means utilizing difference insolubility and means utilizing difference in adsorptive affinity forvarious resins can be used independently, in appropriate combination, orrepeatedly. Specifically, ion-exchange chromatography, chromatographyusing a nonionic adsorptive resin, gel-filter chromatography,chromatography using an adsorbent such as activated carbon, alumina orsilica gel, high-speed liquid chromatography, various other types ofliquid chromatography, crystallization, vacuum concentration,freeze-drying and other such means can be used independently, inappropriate combination, or repeatedly.

[0088] When used as a cell cycle inhibitor, anti-fungal, ortumor-growth-inhibiting compound, the compound of Formula (I) can beadministered by either oral or a non-oral pathways. When administeredorally, it can be administered in capsule, tablet, granule, spray,syrup, or other such form. When administered non-orally, it can beadministered as an aqueous suspension, an oily preparation or the likeor as a drip, suppository, salve, ointment or the like, whenadministered via injection, subcutaneously, intreperitoneally,intravenously, intramuscularly, or the like. Similarly, it may beadministered topically, rectally, or vaginally, as deemed appropriate bythose of skill in the art for bringing the compound of the inventioninto optimal contact with a tumor, thus inhibiting the growth of thetumor. Local administration at the site of the tumor is alsocontemplated, either before or after tumor resection, as are controlledrelease formulations, depot formulations, and infusion pump delivery.

[0089] To formulate the compound of Formula (I) as a cell cycleinhibitor or tumor-growth-inhibiting compound, known surface activeagents, excipients, smoothing agents, suspension agents andpharmaceutically acceptable film-forming substances and coatingassistants, and the like may be used. Preferably alcohols, esters,sulfated aliphatic alcohols, and the like may be used as surface activeagents; sucrose, glucose, lactose, starch, crystallized cellulose,mannitol, light anhydrous silicate, magnesium aluminate, magnesiummethasilicate aluminate, synthetic aluminum silicate, calcium carbonate,sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethylcellulose, and the like may be used as excipients; magnesium stearate,talc, hardened oil and the like may be used as smoothing agents; coconutoil, olive oil, sesame oil, peanut oil, soya may be used as suspensionagents or lubricants; cellulose acetate phthalate as a derivative of acarbohydrate such as cellulose or sugar, or methyiacetate-methacrylatecopolymer as a derivative of polyvinyl may be used as suspension agents;and plasticizers such as ester phthalates and the like may be used assuspension agents. In addition to the foregoing preferred ingredients,sweeteners, fragrances, colorants, preservatives and the like may beadded to the administered formulation of the compound of the invention,particularly when the compound is to be administered orally.

[0090] The cell cycle inhibitor and antitumor agent of the invention maybe orally or non-orally administered to a human patient in the amount ofabout 0.001 mg/kg/day to about 10,000 mg/kg/day of the activeingredient, and more preferably about 0.1 mg/kg/day to about 100mg/kg/day of the active ingredient at, preferably, one time per day or,less preferably, over two to about ten times per day. Alternatively andalso preferably, the compound of the invention may preferably beadministered in the stated amounts continuously by, for example, anintravenous drip. Thus, for the example of a patient weighing 70kilograms, the preferred daily dose of the active anti-tumor ingredientwould be about 0.07 mg/day to about 700 grams/day, and more preferable,7 mg/day to about 7 grams/day. Nonetheless, as will be understood bythose of skill in the art, in certain situations it may be necessary toadminister the anti-tumor compound of the invention in amounts thatexcess, or even far exceed, the above-stated, preferred dosage range toeffectively and aggressively treat particularly advanced or lethaltumors.

[0091] In the case of using the cell cycle inhibitor of the invention asa biochemical test reagent, the compound of the invention inhibits theprogression of the cell cycle when it is dissolved in an organic solventor hydrous organic solvent and it is directly applied to any of variouscultured cell systems. Usable organic solvents include, for example,methanol, methylsulfoxide, and the like. The formulation can, forexample, be a powder, granular or other solid inhibitor, or a liquidinhibitor prepared using an organic solvent or a hydrous organicsolvent. While a preferred concentration of the compound of theinvention for use as a cell cycle inhibitor is generally in the range ofabout 1 to about 100 μg/ml, the most appropriate use amount variesdepending on the type of cultured cell system and the purpose of use, aswill be appreciated by persons of ordinary skill in the art. Also, incertain applications it may be necessary or preferred to persons ofordinary skill in the art to use an amount outside the foregoing range.

[0092] From a pharmaceutical perspective, certain embodiments providemethods for preventing or treating fungal infections and/or a pathogenicfungus in a subject, involve administering to the subject a compositionincluding a phenylahistin or its analog, for example, administering thephenylahistin or its analog in an amount and manner which provides theintended antifungal effect.

[0093] Other embodiments include the treatment or prevention ofinfection in a patient by a pathogenic fungus such as those listed aboveor referred to below.

[0094] Another embodiment relates to the treatment or prevention ofinfection in a patient by a pathogenic fungus which is resistant to oneor more other antifungal agents, especially an agent other thanphenylahistin or its analog, including e.g. amphotericin B or analogs orderivatives thereof (including 14(s)-hydroxyamphotericin B methyl ester,the hydrazide of amphotericin B with 1-amino-4-methylpiperazine, andother derivatives) or other polyene macrolide antibiotics, including,e.g., nystatin, candicidin, pimaricin and natamycin; flucytosine;griseofulvin; echinocandins or aureobasidins, including naturallyoccurring and semi-synthetic analogs; dihydrobenzo[a]napthacenequinones;nucleoside peptide antifungals including the polyoxins and nikkomycins;allylamines such as naftifine and other squalene epoxidease inhibitors;and azoles, imidazoles and triazoles such as, e.g., clotrimazole,miconazole, ketoconazole, econazole, butoconazole, oxiconazole,terconazole, itraconazole or fluconazole and the like. For additionalconventional antifungal agents and new agents under deveopment, see e.g.Turner and Rodriguez, 1996 Current Pharmaceutical Design, 2:209-224.Another embodiment involves the treatment or prevention of infection ina patient by a pathogenic fungus in cases in which the patient isallergic to, otherwise intolerant of, or nonresponsive to one or moreother antifungal agents or in whom the use of other antifungal agents isotherwise contra-indicated. Those other antifungal agents include, amongothers, those antifungal agents disclosed above and elsewhere herein.

[0095] In the foregoing methods for treatment or prevention, aphenylahistin or its analog, is administered to the subject in aneffective antifungal amount.

[0096] Other embodiments relate to the treatment or prevention ofinfection by a pathogenic fungus in a patient by administration of aphenylahistin or its analog, in conjunction with the administration ofone or more other antifungal agents, including for example, any of thepreviously mentioned agents or types of agents (e.g. in combination withtreatment with amphotericin B, preferably in a lipid or liposomeformulation; an azole or triazole such as fluconazole, for example; anaureobasidin; dihydrobenzo[alnapthacenequinone; or an echinocardin) aswell as with a different phenylahistin or its analog.

[0097] The phenylahistin or its analog may be administered before, afteror at the same time the other antifungal agent is administered. Incertain embodiments, the combination therapy will permit the use ofreduced amounts of one or both antifungal components, relative to theamount used if used alone.

[0098] Still other embodiments relate to administration of aphenylahistin or its analog to a subject for the treatment or preventionof infection by a pathogenic fungus, where the subject isimmunosuppressed or immunocompromised, e.g. as the result of geneticdisorder, disease such as diabetes or HIV or other infection,chemotherapy or radiation treatment for cancer or other disease, ordrug- or otherwise induced immunosuppression in connection with tissueor organ transplantation or the treatment of an autoimmune disorder.Where the patient is being or will be treated with an immunosuppressiveagent, e.g., in connection with a tissue or organ transplantation, aphenylahistin or its analog may be co-administered with theimmunosuppressive agent(s) to treat or prevent a pathogenic fungalinfection.

[0099] Another aspect of this invention is the treatment or preventionof infection by a pathogenic fungus in a patient infected, or suspectedof being infected, with HIV, by administration of an antifungalphenylahistin or its analog, in conjunction with the administration ofone or more anti-HIV therapeutics (including e.g. HIV proteaseinhibitors, reverse transcriptase inhibitors or anti-viral agents). Thephenylahistin or its analog may be administered before, after or at thesame time as administration of the anti-HIV agent(s).

[0100] Another aspect of this invention is the treatment or preventionof infection by a pathogenic fungus in a patient by administration of anantifungal phenylahistin or its analog, in conjunction with theadministration of one or more other antibiotic compounds, especially oneor more antibacterial agents, preferably in an effective amount andregiment to treat or prevent bacterial infection. Again, thephenylahistin or its analog may be administered before, after or at thesame time as administration of the other agent(s).

[0101] Pathogenic fungal infections which may be treated or prevented bythe disclosed methods include, among others, Aspergillosis, includinginvasive pulmonary aspergillosis; Blastomycosis, including profound orrapidly progressive infections and blastomycosis in the central nervoussystem; Candidiasis, including retrograde candidiasis of the urinarytract, e.g. in patients with kidney stones, urinary tract obstruction,renal transplantation or poorly controlled diabetes mellitus;Coccidioidomycosis, including chronic disease which does not respondwell to other chemotherapy; Cryptococcosis; Histopolasmosis;Mucormycosis, including e.g. craniofacial mucormycosis and pulmonarymucormycosis; Paracoccidioidomycosis; and Sporotrichosis. It should benoted that administration of a composition comprising an antifungalamount of one or more phenylahistin or its analogs may be particularlyuseful for treating or preventing a pathogenic fungal infection in amammalian subject where the fungus is resistant to one or more otherantifungal therapies, or where the use of one or more other antifungaltherapies is contraindicated, e.g., as mentioned above.

[0102] Antifungal pharmaceutical compositions containing at least oneantifungal phenylahistin or its analog, are also provided for use inpracticing the disclosed methods. Those pharmaceutical compositions maybe packaged together with an appropriate package insert containing,inter alia, directions and information relating to their antifungal use.Pharmaceutical compositions are also provided which contain one or morephenylahistin or its analog together with a second antifungal agent.

Methods of Treating Fungal Infections

[0103] Certain embodiments disclosed herein relate to methods fortreating or preventing a pathogenic fungal infection, including forexample Aspergillosis, including invasive pulmonary aspergillosis;Blastomycosis, including profound or rapidly progressive infections andblastomycosis in the central nervous system; Candidiasis, includingretrograde candidiasis of the urinary tract, e.g. in patients withkidney stones, urinary tract obstruction, renal transplantaion or poorlycontrolled diabetes mellitus; Coccidioidomycosis, including chronicdisease which does not respond well to other chemotherapy;Cryptococcosis; Histopolasmosis; Mucormycosis, including e.g.craniofacial mucormycosis and pulmonary mucormycosis;Paracoccidioidomycosis; and Sporotrichosis. The methods may involveadministering at least one antifungal phenylahistin or its analog, asdescribed above, to a human subject such that the fungal infection istreated or prevented. In certain embodiments the phenylahistin or itsanalog may be administered in conjunction with administration of one ormore non-phenylahistin or its analog antifungal agents such asamphotericin B, or an imidazole or triazole agent such as thosementioned previously.

[0104] The pathogenic fungal infection may be topical, e.g., caused by,among other organisms, species of Candida, Trichophyton, Microsporum orEpiderinophyton or mucosal, e.g., caused by Candida albicans (e.g.thrush and vaginal candidiasis). The infection may be systemic, e.g.,caused by Candida albicans, Cryptococcus neoformans, Aspergillusfumigatus, Coccidiodes, Paracocciciodes, Histoplasma or Blastomyces spp.The infection may also involve eumycotic mycetoma, chromoblastomycosis,cryptococcal meningitits or phycomycosis.

[0105] Further embodiments relate to methods for treating or preventinga pathogenic fungal infection selected from the group consisting ofCandida spp. including C. albicans, C. tropicalis, C. kefyr, C. kruseiand C. galbrata; Aspergillus spp. including A. fumigatus and A. flavus;Cryptococcus neoibrmans; Blastomyces spp. including Blastomycesdermatitidis; Pneumocvstis carinii; Coccidioides immitis; Basidiobolusranarum; Conidiobolus spp.; Histoplasma capsulatum; Rhizopus spp.including R. oryzae and R. microsporus; Cunninghamella spp.; Rhizoniucorspp.; Paracoccidioides brasiliensis; Pseudallescheria boydii;Rhinosporidium seeberi; and Sporothrix schenckii. Again, the method mayinvolve administering a non-immunosuppressive antifungal phenylahistinor its analog to a patient in need thereof such that the fungalinfection is treated or prevented without inducing an untowardimmunosuppressive effect.

[0106] Further embodiments relate to methods for treating or preventinga pathogenic fungal infection which is resistant to other antifungaltherapy, including pathogenic fungal infections which are resistant toone or more antifungal agents mentioned elsewhere herein such asamphotericin B, flucytosine, one of the imidazoles or triazoles(including e.g. fluconazole, ketoconazole, itraconazole and the otherpreviously mentioned examples). The methods may involve administering tothe patient one or more antifungal phenylahistin or its analog, in anamount and dosing regimen such that a fungal infection resistant toanother antifungal therapy in the subject is treated or prevented.

[0107] Further embodiments relate to methods for treating or preventinga pathogenic fungal infection in a patient who is allergic to,intollerant of or not responsive to another antiftingal therapy or inwhom the use of other antifungal agents is otherwise contra-indicated,including one or more other antifungal agents mentioned elsewhere hereinsuch as amphotericin B, flucytosine, one of the imidazoles or triazoles(including e.g. fluconazole, ketoconazole, itraconazole and the otherpreviously mentioned examples). The methods may involve administering tosuch patient one or more antifungal phenylahistin or its analog, in anamount such that a fungal infection is treated or prevented.

Packaged Phenylahistin or its Analogs

[0108] Certain embodiments relate to packaged phenylahistin or itsanalogs, preferably packaged nonimmunosuppressive antifungalphenylahistin or its analogs, which term is intended to include at leastone phenylahistin or its analog, as described above, packaged withinstructions for administering the phenylahistin or its analog(s) as anantifungal agent without causing a untoward immunosuppressive effectswithin a human subject. In some embodiments, the non-immunosuppressiveantifungal phenylahistin or its analog is a member of one of thepreferred subsets of compounds described above. The phenylahistin or itsanalog can be packaged alone with the instructions or can be packagedwith another phenylahistin or its analog, raparnycin or anotheringredient or additive, e.g., one or more of the ingredients of thepharmaceutical compositions. The package can contain one or morecontainers filled with one or more of the ingredients of thepharmaceutical compositions. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceutical orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

[0109] The following examples are meant to describe the preferred modesof making and using the invention, i.e., of isolating, preparing,characterizing, and using certain preferred embodiments of theinvention. Variations in the details of the particular methods employedand in the precise chemical compositions obtained will undoubtedly beappreciated by those of skill in the art.

EXAMPLE 1 Production, Isolation and Purification of RacemicPhenylahistin (PLH)

[0110] During the course of screening for new cell cycle inhibitors, seeRoberge, M.; Tudan, C; Hung, S. M. F.; Harder, K. W.; Jirik, F. R.;Anderson, H. Cancer Res. 1994, 54, 6115, a novel compound NSCL-96F037was found in the culture broth of Aspergillus ustus NSC-F038. seeFukumoto; K.; Asari, T.; Harada, T. Japanese Patent P409188749, Sep. 4,1996 (Japanese). A. ustus NSC-F038 and NSC-F037 fungal strains weredeposited with the National Institute of Bioscience andHuman-Technology, Japan, Agency of Industrial Science and Technology,Ministry of International Trade and Industry, A. ustus NSC-F037 wasdesignated FERM P-15829 and A. ustus NSC-F038 was designated FERMP-15830. The structure and biological activity of the compound initiallydesignated NSCL-96F037 and of several of its derivatives were determinedand are herein described. The compound initially designated NSCL-96F037is herein also termed “phenylahistin” (PLH), and has the followingstructure:

[0111] A. Production, Isolation and Purification of PLH from Aspergillusustus NSC-F037

[0112] In each of 18 mm-diameter, 180 mm-length test tubes, 5 ml ofsterilized production medium (production medium: glucose 5 g/l, glycerin20 ml/l, cotton seed lees 20 g/l, yeast extract 2 g/l, sodium chloride2.5 g/l, calcium carbonate 4 g/l(pH6.5)) was placed. The number ofso-prepared test tubes was 1,700 (total culture medium: 8.5 liter). Eachtest tube was inoculated with 50 μl of Aspergillus ustus NSC-F037 fungussuspension prepared by suspending conidia of the strain in sterilizedwater and the culture broth was incubated for 5 days at 27° C. underreciprocal shaking (260 rpm). The culture broth in each test tube wasadded with 10 ml. of acetone and was extracted for one (1) day at roomtemperature. Impurities were then removed by filtration and the filtrate(approximately 20 liters) was vacuum-concentrated to distill off theacetate. Nine liters of ethyl acetate was added to the remaining waterlayer to effect extraction. The ethyl acetate layer was de-watered withsodium sulfate. Vacuum concentration and drying were conducted to obtaina solid which was subjected to silica gel open column chromatography(chloroform-methanol system) to isolate the antitumor activity fraction.This fraction was vacuumed-concentrated and dried to a solid, dissolvedin a small amount of 70% methanol, subjected to high performance liquidchromatography using a Senshu Pack ODS-5251-SS 20 mm-diameter×250 mm(Senshu Science Kabushiki Kaisha) to conduct purification with a mobilephase: methanol/water 7/3 condition and to isolate a fraction exhibitingantitumor activity. This fraction was vacuum-concentrated and dried to asolid, subject to high performance liquid chromatography using a SenshuPack Aquaseal SS5251(60) 20 mm-diameter×250 mm (Senshu Science KabushikiKaisha) to conduct purification with a mobile phase: chloroform/methanol95/5 and to isolate the antitumor activity fraction. This fraction wasvacuum-concentrated to obtain a transparent oily substance. This wassuspended in a small amount of benzene and the result was vacuumconcentrated and dried to a solid, thereby yielding 25 mg ofNSCL-96F037, also herein termed phenylahistin (PLH).

[0113] B. Production, Isolation and Purification of PLH from Aspergillusustus NSC-F038

[0114] The same steps as above were conducted except Aspergillus ustusNSC-F038 was used instead of Aspergillus ustus NSC-F037. It wasdetermined that the ability of Aspergillus ustus NSC-F038 to produce theantitumor substance NSCL-95F037 was approximately four times that ofNSC-F037. Specifically, from NSC-F037, approximately 2.9 mgphenylahistin was purified per liter of culture medium, while fromNSC-F038, approximately 11.8 mg of phenylahistin was purified per literof culture medium.

[0115] The production of phenylahistin by A. ustus NSC-F038 was alsodetermined to be related to the conidia formation of the fungus.Therefore, the fungus was cultured on an agar medium containing glucose(0.5%), glycerol (2%), yeast extract (0.2%), Pharmamedia (TradersProtein) (2%), NaCl (0.25%) and agar (1.5%), adjusted to pH 6.5 beforesterilization,. This medium was suitable for the conidia formation at28° C. for 8 days. The cultured agar media (200 plates; 4L) were thenextracted with ethyl acetate. The extract was initially subject tosilica gel column chromatography under an ethyl acetate-acetone stepwisegradient, followed by a second silica gel column and eluted with 2%methanol in chloroform. The fractions that exhibited cell cycleinhibitory activity were collected and evaporated, then dissolved inethyl acetate and left two days at room temperature. The activesubstance was precipitated to yield 330 mg of a white amorphous powderwith the following physical and spectroscopic characteristics: meltingpoint: 233-236° C.; [α]_(D) ²²+123° (c=0.13, MeOH); UV (MeOH) 8 max(log,): 202 (4.38), 233 sh (4.05), 320 (4.43); IR (KBr pellet) <max:3440, 3240, 1670, 1640, 1440 cm⁻¹; HR-FAB-MS (m/z) 350.1741 (M⁺, calcd.for C₂₀H₂₂N₄0₂: 350.1743).

[0116] The fungus A. ustus NSC-F038 was, alternatively, cultured is 20ml of a culture medium comprising 0.5% glucose, 2% glycerin, 0.2% yeastextract, 2% cotton seed lees, 0.25% sodium chloride 2.5 g/l and 1.5%agar (pH6.5) to form a planar medium in a 9 cm-diameter Petri dish. Theculture medium was inoculated at 5 points with Aspergillus ustusNSC-F038 and incubated in the dark for 7 days at 26° C. to producespores. The spores were harvested into 5 ml of sterilized water havingTween 20 added to a concentration of 0.05%, thereby obtaining a sporesuspension. Each of 400 Petri dishes containing 20 ml of the sameculture medium was inoculated with 0.1 ml of the spore suspension.Incubation was conducted in the dark at 26° C. for 8 days. The cultureswere comminuted with a mixer, 8 liters of ethyl acetate were added, andthe mixture left standing 2 days before extraction. The recovered ethylacetate was then vacuum-concentrated to obtain 15 g of a brown syrup.The syrup was dissolved in 20 ml of ethyl acetate, and the solution wassubject to a silica gel column (bed volume: 8 cm diameter×20 cm length)chromatography under ethyl acetate and eluted with acetone ethyl acetate(1:5). The eluate was fractionated in 500 ml lots in the order eluted.The active compound was eluted in the fifth to tenth lots. These eluteswere then vacuum-concentrated to afford a dark-brown powder in a totalamount of approximately 4.65 g. The dark-brown powder was then dissolvedin 10 ml of chloroform and the solution was subject to silica gel column(bed volume: 5 cm diameter×30 cm length) chromatography under ethylacetate and eluted first with 500 ml chloroform and then with methanolin chloroform (1:50).

[0117] The active compound was eluted with methanol in chloroform (1:50)to obtain a brown powder in a total amount of approximately 1.05 g. Thebrown powder was then thoroughly mixed with ethyl acetate and left tostand 2 days to precipitate out approximately 628 mg of a white powder(A) containing the active compound. A portion of the white powder (A)was hydrolyzed. The so-obtained phenylalanine was analyzed by highperformance liquid chromatography using a chiral column. The presence ofR-configuration and S-configuration phenylalanine was then confirmed ina standard manner, and it was determined that the white powder (A) was aracemic mixture of the enantiomers of phenylahistin.

[0118] Another portion of the white powder (A) was dissolved in ethylacetate containing a small amount of methanol and left to stand for 7days. As a result a light yellow crystalline solid 187 mg of a purifiedspecimen of (+)-phenylahistin was obtained. These specimens were used inthe following four examples, EXAMPLES 2 through 5, as therein described,to verify the cell-proliferation, antimicrobial, cell-cycle inhibitingeffects of the compound of the invention.

EXAMPLE 2 Concentration of Phenylahistin (PLH) Effective in InhibitingCell Proliferation

[0119] Into each well of a 96-well microtiter plate, 100 μl of A-549cells derived from human lung cancer prepared to 10⁵ cells/ml in aculture medium obtained by adding 10% bovine fetus serum to EMEM culturemedium (Nissui Seiyaku Co., Ltd.) having antitumor effect against A-549cells derived from human lung cancer was placed. Methanol solution ofracemic NSCL-96F037 obtained as in Example 1 was added to the wells ofthe uppermost row, specimens were diluted by the half-log dilutionmethod and added, and the plate was incubated in a carbon dioxide gasincubator at 37° C. for 48 hours. The result was added in lots of 10 μlwith MTT reagent (3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2H-tetrabromide) (1 mg/ml•PBS), followed by incubation in a carbon dioxide gasincubator at 37° C. for 6 hours. The culture medium was discarded andthe crystal of produced in the cells was dissolved in 100 μl/well ofdimethylsulfoxide. Absorption of 595 nm light was then measured with amicroplate reader. By comparing the light absorptions of the untreatedcells to that of cells treated with a specimen of a known concentration,the specimen concentration that inhibited cell proliferation 50% (IC₅₀)was calculated, thus obtaining an IC₅₀=0.3 μg/ml.

EXAMPLE 3 Antimicrobial Test of Phenylahistin (PLH)

[0120] Using colibacillus strain JM109 as a gram-negative bacterium,Bacillus natto as a gram-negative bacterium and Aspergillus nigerIFO6341 as a mold, the following antimicrobial test was conducted by thefilter paper agar flat plate method, wherein 100 μg of specimen wasplaced on 9 mm filter paper, dried in air, and standard agar culturemedium was used for bacteria and potato dextrose agar culture medium formold. No antimicrobial activity was observed. An antimicrobial test wasfurther conducted by the liquid culture medium dilution method using theyeast strain Saccharomyces cerevisise HF7C. Again, no antimicrobialactivity was observed, indicating that the racemic NSCL-96F037(phenylahistin) obtained in EXAMPLE 1 had high animal cell-specificproliferation inhibiting activity.

EXAMPLE 4 Cell Cycle Inhibiting Activity of Phenylahistin (PLH)

[0121] Cell strain A431 derived from human lung cancer was used. EMEMculture medium containing 10% bovine fetus serum and 1% MEM nonessentialamino acid solution (SIGMA M2025) was used to incubate A431 cells at 37°C. in an incubator saturated with 5% carbon dioxide gas and water vapor.The refined specimen of racemic phenylahistin obtained in EXAMPLE 1 wasadded to the cells in the log-growth phase and progression of the cellcycle was analyzed by flow cytometer and microscopic observation. Theresults, shown in Table 1, indicate that this phenylahistin was usefulas a cell cycle inhibitor. TABLE 1 Cell cycle inhibiting activityConcentration (μg/ml) Inhibiting effect 0.5 Absent 1.0 Absent 2.0 Slight4.0 Slight 8.0 Present 16.0 Present 32.0 Present

EXAMPLE 5 Cell Proliferation Inhibiting Effect of Phenylahistin (PLH)

[0122] K-562, human chronic myelogenic leukemia cells were cultured inRPMI164 culture medium (containing 10% bovine fetus serum) and A-431human pudendal epithelial squamous cancer cells were cultured in DMEMculture medium, containing 10% bovine fetus serum. To these a continuousdilution series of the (+)-phenylahistin obtained in EXAMPLE 1 wasadded. After 48-hours incubation, a MTT reagent was added to measuregrowth. The human chronic myelogenic leukemia cells exhibited an IC₅₀ of13.3 μg/ml, while the human pudendal epithelial squamous cancer cellsexhibited an IC₅₀ of 2.9 μg/ml, indicate that (+)-phenylahistin wasuseful as an antitumor agent.

EXAMPLE 6 Characterization and Resolution of Enantiomers ofPhenylahistin (PLH)

[0123] A. Racemic Phenylahistin

[0124] Phenylahistin (PLH) had the molecular formula, C₂₀H₂₂N₄0₂, whichwas determined by HR-FAB-MS (mz found: 350.1741 (M⁺), calcd. forC₂₀H₂₂N₄0₂: 350.1743). In the IR spectrum, the absorption at 3440 cm⁻¹corresponds to the N—H group, and strong absorptions at 1670 and 1640(cm⁻¹ indicated the existence of amide groups. These findings togetherwith the absence of the amide II band near 1550 cm⁻¹ in the spectrumsuggested the presence of the diketopiperazine system in phenylahistin,according to the technique described in Steyn, P. S. Tetrahedron 1973,29, 107, which was also supported by the negative ninhydrin reaction.

[0125] The ¹³C-NMR spectrum of phenylahistin, with atomic positions asindicated in Structure (IV), summarized in the first column of Table 2,showed 17 resolved peaks with three overlapping carbon signals. Themultiplicity of these peaks was determined by analysis of its DEPTspectra. The ¹H-NMR spectrum displayed 22 proton signals, listed in thesecond column of Table 2, including three exchangeable protons. All bondconnections between proton and carbon were interpreted by ¹H-¹³C COSY.TABLE 2 Table ¹³C and ¹H NMR Assignment of Phenylahistin in CDCl₃ (IV)

Positions δ C* δ H** 1 (NH) — 9.48(1H, br s) 2 132.56 d 7.55(1H, s) 4132.18 s — 5 136.83 s — 6 105.62 d 6.88(1H, s) 7 123.64 — 8 (NH) —12.08(1H, br s) 9 164.73 s — 10 57.14 d 4.35(1H, ddd, J=10, 4, 3#Hz) 11(NH) — 5.82(1H, br s) 12 159.94 s — 13 41.23 t 2.95(1H, dd, J=14, 10Hz)3.49(1H, dd, J=14, 4Hz) 14 135.45 s — 15, 19 129.52 d 7.25(2H, d, J=7Hz)16, 18 129.07 d 7.33(2H, t, J=7Hz) 17 127.45 d 7.27(1H, t, J=7Hz) 2037.61 s — 21 144.66 d 6.02(1H, dd, J=18, 11Hz) 22 113.29 t 5.13(1H, d,J=18Hz) 5.17(1H, d, J=11Hz) 23, 24 27.97 q 1.49(6H, s)

[0126] The ¹H NMR and ¹H -¹H COSY spectra revealed only the presence offour partial structures: a monosubstituted benzene ring, methyleneprotons being coupled to a methine proton, which was also coupled to anexchangeable proton, one geminal methyl group and a vinyl group. Sincequaternary carbons prevented constructing further partial structures,the PFG-HBMC spectrum were t measured. A partial structure wasdetermined on the basis of the long-range correlations of C-9, C-13, andC-14, which were observed in the spectrum. The carbon chemical shift ofC-9 (* 164.73) indicated that it was a carbonyl carbon. Therefore, thatpartial structure was determined to be a phenylalanine residue. In thesame manner, a second partial structure was determined from thecorrelations of C-5, C-20, C-21, C-23 and C-24, which showed anisoprenyl group binding to quaternary sp² carbon (* 136.83). Thediketopiperazine ring was also constructed from the interpretation ofcorrelations between respectively, C-7 and 11-H, C-10 and 8-H, C-12 and8-H, and C-12 and 10-H in the PFG-HMBC spectrum. The dehydrohistidinemoiety was estimated from the remaining three carbons, two nitrogens andthree hydrogens, and this structure was supported by correlation signalsof C-4, C-5, C-7, and C-12. The correlation signal between C-5 and H-6indicated that the isoprenyl group was placed in the C-5 of theimidazole ring. This structure was confirmed by analysis of thePFG-¹⁵N-HMBC spectrum. The nitrogen residues were assigned as follows:N1 (* 159); N3 (* 253); N8 (* 133); N11(* 109) (¹⁵N-formamide internalstandard at * 112.4 ppm).

[0127] From these data, the planar structure of phenylahistin wasestablished. The stereochemistry of the C-6—C-7 double bond wassuggested to be Z by the low field shift of 8-H (NH: * 12.08), which wasfurther explained by hydrogen-bonding between the 8-H proton and N-3 ofthe imidazole ring, as in the case of aurantiamine.

[0128] It was also determined that phenylahistin has a chiral center atthe C-10 position. Chiral HPLC analysis of the phenylalanine wasconducted according to methods described in Larsen, T. O et al.,Phytochemistry (1992) 31, 1613, under the following conditions: HPLCconditions: column; Crownpak CR(+) N4.0×150 mm (Daicel ChemicalIndustries, Ltd.), mobile phase; H₂O (pH 2, adjusted with perchloricacid), flow rate; 0.8 ml/min, detector; UV 8 200 nm. Phenylalanine wasobtained from the acidic hydrolysate of the amorphous white powder ofphenylahistin, see Yamazaki, M. et al., Tetrahedron Lett. (1975) 27. Thephenylalanine obtained from the hydrolysate of phenylahistin wasidentified using an amino acid analyzer and by measurement of EI-MS[(m+H)⁺:166.2]); chrial HPLC indicated that the phenylahistin was amixture of enantiomers in the ratio of approximately R:S=3.1. In orderto examine the biological activity of each enantiomer, chiral resolutionwas carried out by chiral HPLC under the following conditions: column;Chiracel OD N4.6×250 mm (Daicel Chemical Industries, Ltd), mobile phase;n-hexane/ethanol=75/25, flow rate; 1.0 ml/min, temperature; 25° C.Absolute configuration was determined by analysis of phenylalanineobtained from (+)-phenylahistin using chiral HPLC.

[0129] B. Single-Crystal X-Ray Diffraction Analysis of Phenylahistin

[0130] A pale yellow crystal having approximately dimension 0.4×0.4×0.8mm was obtained from the ethyl acetate solution of phenuylahistin. AllX-ray measurements were made on a Rigaku AFC7R diffractometer withgraphite monochromated CuKα radia and a rotating anode generator. Cellconstants and an orientation matrixed for data collection were obtainedfrom a least-squares refinement using the setting angles of 25 carefullycentered reflections in the range of 77.59<26<79.03°. These crystal dataare summarized in Table 3. TABLE 3 Crystal data of (−)-phenylahistinEmpirical formula C₂₀H₂₂N₄O₂ Formula weight 350.42 Crystal systemtetragonal Lattice parameters: a = 15.3509(7) Å o = 8.309(2) Å V =1958.0(2) Å³ Z = 4 Space group P4₂(#77) D calc. 1.189 g/cm³ μ (CuKα)6.37 cm⁻¹

[0131] Of the 2229 reflections which were collected, 2064 were unique(R_(int)=0.019). The intensities of the three representative reflectionswere measured after every 150 reflections. No decay collection wasapplied. The linear absorption coefficient: μ for CuKα is 6.37 cm⁻¹.Azimuthal scans of several reflections indicated no need for anabsorption correction. The data were corrected for Lorentz andpolarization effects. A correction for secondary extinction was applied.The structure was solved by direct methods, see SAPA91: Fan Hai-Fu(1991). Structure Analysis Programs with Intelligent Control, RigakuCorporation, Tokyo, Japan, and expanded using Fourier techniques, seeDIRDIF94: Beurskens, P. T. et al. (1994) the DIRDIF94 program system,Technical Report of the Crystallography Laboratory, University ofNijmegen, The Netherlands. Nonhydrogen atoms except for C22 were refinedanisotropically, while C22 was refined isotropically. Some hydrogenatoms were refined isotropically, the est included in fixed positions.The final cycle of full-matrix least-squares refinement was based on1724 observed reflections (I>1.50σ(I)) and 242 variable parameters andconverged (largest parameter shift was 0.30 times its esd) withunweighted and weighted factors of :R=0.057, R_(w)=0.067. The maximumand minimum peaks on the final difference Fourier map corresponded to0.20 and −0.12 e⁻/A⁸, respectively. All calculations were performedusing the teXscan crystallographic software package of MolecularStructure Corporation.

[0132] The stereochemistry of C6—C7 double bond was confirmed to be Z,and the hydrogen bonding between 8(N)—H and N-3 was observed asspeculated from low field-shift of 8(N)—H (δ12.08 ppm) in NMR study.Since the absolute configuration of C-10 position could not bedetermined by this X-ray analysis, we analyzed the configuration ofphenylalanine which was obtained from acidic hydrolysate of thecrystalline sample using chiral HPLC. The phenylalanine obtained fromthe crystalline sample was identical with the authentic sample ofD-(+)-phenylalanine (R-configuration). Therefore, the absoluteconfiguration of this crystalline sample of phenylahistin wasestablished to be R.

[0133] Other derivatives of phenylahistin can be synthesized using theforegoing techniques or others well known organic synthesis techniques.

EXAMPLE 7 Synthesis and Physical Characterization of PhenylahistinDerivatives

[0134] A. Synthesis of Various Derivatives of Phenylahistin

[0135] Structural derivatives of phenylahistin were synthesized from theresolved enantiomers according to the following reaction schemes. InReaction Scheme 1, shown in FIG. 1, the enantioners of phenylahistinwere resolved, as described in Example 6, and each was subject topalladium-based catalytic reduction conditions for 2 hours, yielding twomono-reduced phenylahistin derivatives herein designated compounds 3 and4. These compounds were in turn subject to palladium-based catalyticreduction conditions for 24 hours, yielding di-reduced phenylahistinderivatives, herein designated compounds 5 and 6.

[0136] In Reaction Scheme 2, as shown in FIG. 2, the synthesis of thephenylahistin derivative designated PLH-C1, and alternatively designatedcompound 9, is described. This reaction scheme treats H₂N-Phe-Gly-OMe, acommercially available di-peptide derivative to yield PLH-C1, aderivative of PLH in which the isoprenyl moiety is replaced with amethyl group. Since partially racemization was occurred (21%), chiralresolution was carried out to obtain optically pure PLH-C1.

[0137] In Reaction Scheme 3, as shown in FIG. 3, the synthesis of(−)-PLH-C1 is described. In the course of synthesizing this compound,another phenylahistin derivative, designated compound 7 is synthesized.

[0138] B. Physical Characteristics

[0139] These above-identified compounds were characterized as follows:

[0140] Compound 3.

[0141] Colorless solid; mp 224-226° C.; [α]_(D) ²⁵−295°(c=0.15, MeOH),UV (MeOH) nm 322 (ε 23300), 231 (ε 8830), 203 (ε 16800); IR (KBr) cm⁻¹3310, 3220, 2970, 1670, 1440; ¹H NMR (270 MHz, CDCl₃) δ 12.09 (br s,1H), 8.98 (br s. 1H), 7.56 (s, 1H), 7.29-7.38 (m, 5H), 6.87 (s, 1H),5.67 (s, 1H), 4.34 (ddd, J=2, 3, 10 Hz, 1H), 3.51 (dd, J=3, 14 Hz, 1H),2.29 (dd, J=10, 14 Hz, 1H), 1.74 (q, J=7 Hz, 2H), 1.40 (s, 6H), 0.74 (t,J=7 Hz 3H); ¹³C NMR (67.5 MHz, CDCl₃) δ 164.6, 159.9, 138.1, 135.5,132.2, 132.1, 129.5 (2C), 129.1 (2C), 127.4, 123.6, 105.4, 57.2, 41.3,36.2, 35.4, 27.9 (2C), 9.2; High-resolution MS m/z 352.1926 (M⁺) (Calcdfor C₂₀H₂₄N₄O₂: 352.1899). Anal. calculated for C₂₀H₂₄N₄O₂: C, 68.16; H,6.86; N, 15.90. Found: C, 68.34, 6.78, 15.82.

[0142] Compound 4:

[0143] Colorless solid; mp 222-223° C; [α]_(D) ²⁵ +284°(c=0.10, MeOH),UV (MeOH) nm 322 (ε 22800), 231 (ε 8720), 203 (ε 16300); IR (KBr) cm⁻¹3310, 3220, 2970, 1670, 1440; ¹H NMR (270 MHz, CDCl₃) δ 12.09 (br s,1H), 8.98 (br s. 1H), 7.56 (s, 1H), 7.29-7.38 (m, 5H), 6.87 (s, 1H),5.67 (s, 1H), 4.34 (ddd, J=2, 3, 10 Hz, 1H), 3.51 (dd, J=3, 14 Hz, 1H),2.29 (dd, J=10, 14 Hz, 1H), 1.74 (q, J=7 Hz, 2H), 1.40 (s, 6H), 0.74 (t,J=7 Hz 3H); ¹³C NMR (67.5 MHz, CDCl₃) δ 164.6, 159.9, 138.1, 135.5,132.2, 132.1, 129.5 (2C), 129.1 (2C), 127.4, 123.6, 105.4, 57.2, 41.3,36.2, 35.4, 27.9 (2C), 9.2; High-resolution MS m/z 352.1932 (M⁺) (Calcdfor C₂₀H₂₄N₄O₂: 352.1899). Anal. calculated for C₂₀H₂₄N₄O₂: C, 68.16; H,6.86; N, 15.90. Found: C, 68.09, 6.87, 15.84.

[0144] Compound 5:

[0145] Colorless solid; mp 224-225° C.; [α]_(D) ²⁵ −96°(c=0.16, MeOH),UV (MeOH) nm 257 (ε 194), 205 (ε 18100), IR (KBr) cm⁻¹ 3380, 3200, 2970,1670, 1440; ¹H NMR (270 MHz, DMSO-d₆) δ 11.55 (br s, 1H), 8.25 (br s1H), 7.86 (br s 1H), 7.41 (s, 1H), 7.29-7.14 (m, 5H), 4.24 (br s, 1H),3.94 (brd, J=11 Hz, 1H), 3.11 (dd, J=14, 4 Hz, 1H), 2.88 (dd, J=14, 5Hz, 1H), 2.82 (dd, J=15, 2 Hz, 1H), 1.45 (q, J=7 Hz, 2H), 1.32 (dd,J=15, 11 Hz, 1H), 1.13 (s, 3H), 1.12 (s, 3H), 0.60 (t, J=7 Hz, 3H); ¹³CNMR (67.5 MHz, DMSO-d₆) δ 166.9 165.4, 135.9, 132.5, 131.2, 130.3 (2C),127.9 (2C), 126.6, 55.7, 54.3, 38.2, 35.0, 34.3, 31.9, 27.6, 27.5, 9.1;High-resolution MS m/z 354.2098 (M⁺) (Calcd for C₂₀H₂₆N₄O₂: 354.2055).Anal. calculated for C₂₀H₂₆N₄O₂: ⅓H₂O: C, 66.64; H, 7.46; N, 15.54.Found: C, 66.75, 7.41, 15.52.

[0146] Compound 6:

[0147] Colorless solid; mp 226-227° C.; [α]_(D) ²⁵ +99°(c=0.10, MeOH),UV (MeOH) nm 257 (ε 174), 205 (ε 18200), IR (KBr) cm⁻¹ 3380, 3200, 2970,1670, 1440; ¹H NMR (270 MHz, DMSO-d₆) δ 11.55 (br s, 1H), 8.25 (br s.1H), 7.86 (br s, 1H), 7.41 (s, 1H), 7.29-7.14 (m, 5H), 4.24 (br s, 1H),3.94 (br d, J=11 Hz, 1H), 3.11 (dd, J=14, 4 Hz, 1H), 2.88 (dd, J=14, 5Hz, 1H), 2.82 (dd, J=15, 2 Hz, 1H), 1.45 (q, J=7 Hz, 2H), 1.32 (dd,J=15, 11 Hz, 1H), 1.13 (s, 3H), 1.12 (s, 3H), 0.60 (t, J=7 Hz, 3H); ¹³CNMR (67.5 MHz, DMSO-d₆) δ 166.9, 165.4, 135.9, 132.5, 131.6, 131.2,130.3 (2C), 127.9 (2C), 126.6, 55.7, 54.3, 38.2, 35.0, 34.3, 31.9, 27.6,27.5, 9.1; High-resolution MS m/z 354.2110 (M⁺) (Calcd for C₂₀H₂₆N₄O₂:354.2055). Anal. calcd for C₂₀H₂₆N₄O₂: ½H₂O: C, 66.09; H, 7.49; N,15.41. Found: C, 65.80, 7.50, 15.30.

[0148] Compound 7:

[0149] Cyclo-Gly-Phe (7). White powder; mp 262-263° C. (decomp.);[α]_(D) ²⁵ +60°(c=0.15, DMSO); UV (MeOH) nm 257 (ε 101), 206 (ε 5770) IR(KBr) cm⁻¹ 3340, 3200, 3060, 1680, 1470, 1340; ¹H NMR (270 MHz,DMSO-d₆)δ 8.16 (br s, 1H), 7.90 (br s. 1H), 7.32-7.15 (m, 5H), 4.07 (br dd, J=7,4 Hz, 1H), 3.35 (dd, J=18, 3 Hz, 1H), 3.10 (dd, J=14, 4 Hz, 1H), 2.88(dd, J=14, 5 Hz, 1H), 2.75 (d, J=18 Hz, 1H); ¹³C NMR (DMSO-d6) δ 167.1,165.7, 136.0, 130.1 (2C), 128.1 (2C), 126.8, 55.5, 43.7, 38.8; Anal.calculated for C₁₁H₁₂N₂O₂: ⅕H₂O: C, 63.57; H, 6.01; N, 13.48. Found: C,63.85, H, 5.86, N, 13.40.

[0150] Compound 8:

[0151] Colorless solid; mp 84-85° C.; [α]_(D) ²⁵ +7.8° (c=0.52, MeOH);UV (MeOH) nm 209 (ε 20400); IR (KBr) cm⁻¹ 1720, 1400, 1380, 1240; ¹H NMR(270 MHz, CDCl₃) δ 7.33-7.26 (m, 3H), 7.08-7.05 (m, 2H), 5.44 (t, J=5Hz, 1H), 4.49 (d, J=19 Hz, 1H), 3.35 (dd, J=14, 5 Hz, 1H), 3.20 (dd,J=14, 5 Hz, 1H), 2.58 (s, 3H), 2.55 (s, 3H), 2.48 (d, J=19 Hz, 1H); ¹³CNMR (67.5 MHz, CDCl₃) δ 171.2, 171.0, 167.9, 166.0, 134.3, 129.7 (2C),129.1 (2C), 128.2, 59.0, 46.0, 38.7, 27.1, 26.8; MS (ESI) m/z 205 (M+H);MS (ESI) m/z 311 (M+Na)⁺; Anal. calculated for C₁₅H₁₆N₂O₄: C, 62.49; H,5.59; N, 9.72. Found: C, 62.50, H, 5.50, N, 9.67.

[0152] Compound 9:

[0153] Colorless solid; mp 285-286° C. (decompose); [α]_(D) ²⁵ −267°(c=0.21 DMSO); UV (MeOH) nm 319 (ε 22800); IR (KBr) cm⁻¹ 3400, 3180,1680, 1450; ¹H NMR (270 MHz, DMSO-d₆) δ 11.50 (br s, 1H), 8.35 (br s,1H), 7.74 (s, 1H), 7.24-7.14 (m, 5H), 6.20 (s, 1H), 4.48 (m, 1H), 3.33(br s, 1H), 3.20 (dd, J=14, 4 Hz, 1H), 2.93 (dd, J=14, 5 Hz, 1H), 2.19(s, 1H); ¹³C NMR (67.5 MHz, DMSO-d₆) δ 164.2, 158.7, 135.6, 134.6,132.3, 130.0 (2C), 128.0 (2C), 127.5, 126.6, 123.4, 101.7, 55.9, 38.7,8.9; High-resolution MS m/z 296.1261 (M⁺) (Calcd for C₁₆H₁₆N₄O₂:296.1273). Anal. calculated for C₁₆H₁₆N₄O₂.⅕H₂O; C, 64.07; H, 5.51; N,18.68. Found: C, 64.39, H, 5.65, N, 18.29.

EXAMPLE 8 Relative Cytotoxic Effects of the Phenylahistin Enantiomers

[0154] The cytotoxic effects of the enantiomers of phenylahistin on P388murine leukemia cells were examined. IC₅₀ values of (−)-phenylahistin(S-configuration) (92.2% e.e.) and (+)-phenylahistin (R-configuration)(97.8% e.e.) were 3.5×10⁻⁷ M and 3.8×10⁻⁵ M, respectively. Judging fromthe content of (−)-phenylahistin in (+)-phenylahistin, (+)-phenylahistinwas considered to have relatively low cytotoxicity to P388 cells.

EXAMPLE 9 Biological Activity of Phenylahistin and its Derivatives

[0155] The biological activity of phenylahistin and its derivatives,synthesized as described in EXAMPLE 7 and established according to themethods described in EXAMPLES 5 and 11, are summarized in Table 4. TABLE4 Comparative Biological Activity of Phenylahistin and its DerivativesIC₅₀ for P388 IC₅₀ for Microtubule Protein Compound Proliferation* (μM)Polymerization** (μM) 1 0.21 25 2 10 >200 3 0.23 30 4 19 >2005 >200 >200 6 >200 >200 7 >200 >200 9 7.5 >200 Cyclo-His-Phe*** >200>200

EXAMPLE 10 Effect of Phenylahistin on the Cell Cycle Progression of P388

[0156] The effect of phenylahistin on the cell cycle progression of P388cells was also investigated using a flow cytometer, as described inKrishan, A., J. Cell Biol. (1975) 66, 188-193. P388 cells in the loggrowth phase were seeded into flasks and cultured for 14 hours, and thenvarious concentrations of phenylahistin were added to each flask. Afteran 8-hour incubation, the cells were harvested and fixed with 50% MeOHat −20° C. overnight. The cells were washed with 30% MeOH and then with10 mM PBS, and treated with 50 :g/ml propidium iodide at 4° C. for 2hours. DNA histograms were obtained using a flow cytometer (CytoACE-300:JASCO).

[0157] (−)-Phenylahistin exhibited cell cycle inhibitory activity at1×10⁻⁶ M, but (+)-phenylahistin (97.8% e.e.) had no effect at 1×10⁻⁵ M.Therefore, the active configuration of phenylahistin was determined tobe S, and it was determined that this configuration most effectivelyinhibited cell cycle progression in the G2/M phase.

EXAMPLE 11 Effects of Phenylahistin on Microtubulin Function

[0158] (−)-Phenylahistin ((−)-PLH) was shown to effect microtubulefunction, specifically via effecting the proliferation, mitosis, andmicrotubule structure of A549 cells (human lung carcinoma), and viainhibiting the in vitro polymerization of bovine brain microtubuleprotein and purified tubulin. In addition, competitive bindingexperiments using colchicine (CLC) and vinblastine (VLB), two typicalantimitotic agents that bind to individual binding sites on tubulin,were used to establish the effect of (−)-PLH on the distinct bindingsites on tubulin.

[0159] A. Materials.

[0160] (−)-PLH was isolated from the agar-culture medium of A. ustusNSC-F038 (See, Kanoh, K. et al.: Bioorg. Med. Chem. Lett. (1997), 7,2847-52, as disclosed in Example 1. CLC was obtained from Sigma (St.Louis, Mo.) and VLB was from Wako Pure Chemicals (Tokyo, Japan). [³H]CLCwas purchased from Du Pont/Boston Nuclear (New England, Mass.) and[³H]VLB was from Amersham (Buckinghamshire, UK). A549 cell line (humanlung carcinoma) was obtained from American Type Culture Collection(Rockville, Md.). A549 cells were cultured in phenol red free EMEMmedium from Nissui Pharmaceuticals (Tokyo, Japan), supplemented with MEMnon-essential amino acids (Sigma) and 10% fetal bovine serum from JRHBiosciences, (Lenexa, Kans.).

[0161] B. Alamar Blue™ Assay.

[0162] Exponentially growing A549 cells were seeded into 96-well tissueculture plates (2×10³ cells/100 μl/well) and cultured for 16 hours.(−)-PLH or CLC was then added to each well at various concentrations,and the cells were cultured for an additional 48 hours. Live cells werecounted using Alamar Blue™ from BioSource International (Camarillo,Calif.) as described in Ahmed, S. A., J. Immunol. Methods, (1994), 170,211-24.

[0163] C. Mitotic Index.

[0164] Exponentially growing A549 cells were seeded into 96-well tissueculture plates (2×10³ cells/100:1/well) and cultured for 16 hours.(−)-PLH or CLC was then added to each well at various concentrations,and the plates were incubated for an additional 24 hours. The number ofcells in mitosis (round cells) and total cells in eight randomlyselected fields were counted under a phase-contrast microscope. In aseries of preliminary studies, it was confirmed that the round cells inthis condition were cells in mitosis by using flow cytometry and Hoechst33258 staining. The mitotic index represented the percentage of roundcells among the total number of cells in the eight selected fields.

[0165] D. Immunocytochemistry.

[0166] Immunocytochemical staining was performed using the methoddescribed by Yahara, I. et al., Cell (1978) 15, 251-259. A549 cells werecultured on glass coverslips and incubated with the test drug for 6hours. The cells were then fixed with 3.7% formaldehyde for 30 min,permeabilized with 0.2% Triton X-100 for 5 min, and incubated with amouse monoclonal antibody against α-tubulin (Calbiochem®, OncogeneResearch Products, Cambridge, Mass.), followed by incubation withfluorescein isothiocyanate-conjugated goat anti-mouse IgG from Cosmo BioCo., (Tokyo, Japan), and the cells were examined under animmunofluorescent microscope.

[0167] E. Microtubule Protein and Tubulin Preparation.

[0168] Microtubule protein was prepared from bovine brain tissue by twocycles of assembly and disassembly according to the method of Tiwari, S.C. et al. Anal. Biochem, (1993), 215, 96-103. Tubulin was purified frommicrotubule protein by phosphocellulose chromatography see Algaier, J.et al. Biochim. Biophys. Acta, (1988), 954, 235-43, and tubulin puritywas evaluated by polyacrylamide gel electrophoresis, as in Laemmli,U.K., Nature, (1970), 277, 680-85. Essentially, nomicrotubule-associated proteins were detected in this preparation.Protein concentrations were determined using the Coomassie® ProteinAssay Reagent from Pierce (Rockford, Ill.).

[0169] F. Polymerization Assay.

[0170] Polymerization of microtubule protein was monitored by anincrease in turbidity at 37° C. in microtubule assembly buffercontaining 100 mM MES, 0.5 mM MgCl₂, 1 mM EGTA and 1 mM GTP. SeeJohnson, K. A. et al., J. Mol. Biol., (1977), 117, 1-31. Polymerizationof purified tubulin was measured using the same method except themicrotubule assembly buffer contained 4 M glycerol. See Lee, J. C. etal., Biochemistry, (1975), 14, 5183-87. Microtubule protein and tubulinpolymerization was initiated by a temperature shift from 0° C. to 37° C.and turbidity was measured on a DU-20 thermocontrolled spectrophotometerfrom Beckman (Fullerton, Calif.) at 360 nm. Drugs were dissolved indimethyl sulfoxide (DMSO), which was used in all experiments at a finalconcentration of 2% (v/v).

[0171] G. Electron Microscopy.

[0172] Microtubule protein (1.5 mg/ml) was polymerized at 37° C. for 20min in the presence of 100 μM (−)-PLH or vehicle (DMSO). A portion ofeach sample was diluted 5-fold with 1% glutaraldehyde in the microtubuleassembly buffer. Samples were then placed on formvar- and carbon-coatedgrids, stained with 2% uranyl acetate, and examined using a JEOLJEM-1200EXII electron microscope JEOL (Tokyo, Japan).

[0173] H. Competition Assay.

[0174] [³H]CLC binding to tubulin was evaluated by the ultrafiltrationmethod of Takahashi, M., et al., Biochim. Biophys. Acta, (1987) 926,215-23, with only slight modifications: bovine brain tubulin (0.2 mg/ml)was incubated in the microtubule assembly buffer with variousconcentrations of [³H]CLC for 20 min at 37° C.; each sample (200 μl) wasapplied to the reservoir of a UFC3LTK00 ultrafiltration unit from NihonMillipore Ltd. (Yonezawa, Japan) and centrifuged at 1,500×g for 4 min atroom temperature to obtain approximately 60 μl of filtrate. Theconcentration of unbound [³H]CLC in the filtrates was determined using aEcoLite™(+) liquid scintillator from ICN Pharmaceuticals Inc. (CostaMesa, Calif.). Specific bound [³H]CLC concentrations were determined byadding an excess (×100) of unlabeled CLC to the reaction mixture.

[0175] For the competition assay, bovine brain tubulin (0.2 mg/ml) wasincubated in the microtubule assembly buffer with 0.5 μM [³H]CLC andvarious concentrations of competitors for 20 min at 37° C. [³H]CLCbinding was measured as described above. DMSO was used as a co-solventat a concentration that did not affect drug binding to tubulin (final 2%v/v). Measurement of [³H]VLB binding was followed by the DEAE-cellulosefilter method of Borisy, G. G., Anal. Biochem. (1972) 373-85, and thecompetition assay was carried out as described for CLC.

[0176] I. Inhibitory Effects of (−)-PLH on Proliferation and Mitosis ofA549 Cells.

[0177] The effects of (−)-PLH on the proliferation and mitosis of A549cells was examined. (−)-PLH inhibited the proliferation of A549 cells ina dose-dependent manner with a 50% inhibitory concentration (IC₅₀) valueof 0.3 μM, indicating that (−)-PLH was 5-fold less potent than CLC underthe assay conditions. However, the mitotic index increased, whichcorrelated with decreased cell proliferation. These results indicatedthat (−)-PLH arrested the cell cycle during mitosis, which subsequentlyreduced cell proliferation, similar to the effect observed for othermitotic inhibitors such as CLC and VLB. See Yoshimatsu, K., et al.,Cancer Res., (1997), 57, 3208-13. The anti-mitotic effect of (−)-PLHseemed to be reversible, because cells that arrested in M phase by(−)-PLH returned to proliferate again following washing of (−)-PLH andreplacement with a fresh medium.

[0178] J. Immunofluorescence Staining of Microtubules in A549 Cells.

[0179] The effects of (−)-PLH on microtubule structure in A549 cellsusing an anti α-tubulin antibody and a secondary antibody conjugatedwith FITC were also investigated. It was determined that (−)-PLHinhibited the microtubule assembly like CLC, but did not hyper-stabilizemicrotubules in a manner similar to that of paclitaxel (Taxol®).

[0180] In control cells, a network of cytoskeletal microtubules wasclearly visible, and mitotic spindles could be observed in mitoticcells. The addition of (−)-PLH resulted in the disappearance of themicrotubule network, and the entire mitotic cell was uniformly stainedwith α-tubulin antibody, whereas mitotic spindles were never seen. Cellsincubated with CLC exhibited a staining profile similar to that of(−)-PLH-treated cells. Paclitaxel, a potent microtubule-stabilizer,produced thick microtubule bundles and multiple bright fluorescent asterformation. See Rowinsky, E. K. et al., J. Natl. Cancer Inst. (1990)82:1247-59. These results suggested that (−)-PLH inhibited microtubuleassembly in A549 cells, but did not stabilize microtubules.

[0181] K. (−)-PLH Effects Microtubule Protein Polymerization andPurified Tubulin.

[0182] The effect of (−)-PLH on the polymerization of microtubuleprotein obtained from bovine brain were also examined. Accordingly, thetime course of the turbidity change in the presence of variousconcentrations of (−)-PLH and CLC was examined. (−)-PLH inhibited the invitro polymerization of microtubule protein in a concentration-dependentmanner, 80 μM of (−)-PLH completely inhibited the polymerization ofmicrotubule protein, while (+)-PLH did not affect microtubule assemblyat concentrations ranging up to a concentration of 200 μM.

[0183] It is reported that VLB, see Luduena, R. F.; J. Biol. Chem.(1984) 259:12890-98, or dolastin 10, see Li, Y., et al, Chem. Biol.Interact., (1994), 93, 175-83 increases the turbidity in microtubuleprotein polymerization at concentrations higher than those required forinhibition. However, (−)-PLH did not increase the turbidity atconcentrations up to 200 μM.

[0184] To determine whether (−)-PLH acts on tubulin directly or onassociated proteins, further tests were performed on the assembly usingphosphocellulose-purified tubulin, which was free of microtubuleassociated proteins. Like microtubule protein, (−)-PLH inhibited tubulinpolymerization in a concentration-dependent manner. The IC₅₀ value ofCLC was 6.6+1.7 μM under the same experimental conditions. These resultsindicated that (−)-PLH was as effective as CLC in inhibiting tubulinpolymerization, and that (−)-PLH acted on tubulin directly rather thanon tubulin-associated proteins.

[0185] The effect of (−)-PLH on polymerization of the microtubuleprotein were also studied by electron microscopy. The control samplecontained singular microtubules with a normal cylindrical structure,whereas a sample treated with 100 μM (−)-PLH did not contain suchstructures. These findings confirmed the turbidity measurements andindicated that (−)-PLH inhibited the polymerization of microtubuleprotein.

[0186] L. (−)-PLH Inhibited CLC Binding to Tubulin.

[0187] To investigate the binding site of (−)-PLH on tubulin,competitive binding studies using [³H]CLC and [³H]VLB were conducted.[³H]CLC and [³H]VLB are antimitotic agents that bind to CLC and VLBbinding sites, respectively. [³H]CLC binding was measured by theultrafiltration method as described above. Under the experimentalconditions, the K_(d) value of CLC to tubulin was 5.3×10⁻⁷M, which is ingood agreement with the previously reported value, see Sherline, P., etal., J. Biol. Chem., (1975), 250, 5481-86. (−)-PLH inhibited CLC bindingto tubulin in a dose-dependent manner, with an estimated K_(i) value forCLC binding of 7.4×10⁻⁶M according to a Dixon plot, assuming that(−)-PLH is the competitive inhibitor of CLC binding to tubulin. VLBslightly enhanced CLC binding to tubulin, likely an effect of thestabilization of VLB on the CLC binding activity of tubulin. See Lacey,E. et al., Biochem. Pharmacol. (1987) 36:2133-38. On the other hand, thebinding of [³H]VLG was not inhibited by (−)-PLH. CLC enhanced and(−)-PLH slightly enhanced [³H]VLG binding to tubulin. These resultssuggested that (−)-PLH binds to the CLC binding site (CLC-site) ontubulin or to a site overlapping the CLC-site.

[0188] (−)-PLH dose-dependently increased the mitotic index in parallelwith inhibition of proliferation of A549 cells, indicating that itprevents cell proliferation by arresting the cell cycle in M phase. Mostantimitotic agents such as CLC and VLB are known to exertanti-microtubule activity, including disruption of the process ofmitotic spindle formation, resulting in cell arrest in mitosis. (−)-PLHalso exhibited an anti-microtubule activity, as evident bydepolymerization of cytoskeletal microtubule in A549 cells treated with(−)-PLH. To investigate the mechanism underlying the anti-microtubuleactivity of this compound, an in vitro polymerization assay using amicrotubule protein and phosphocellulose-purified tubulin from bovinebrain was performed. (−)-PLH inhibited polymerization of bothmicrotubule protein and purified tubulin, suggesting that (−)-PLHdirectly acted on tubulin, rather than interacting with microtubuleassociated proteins. Although (−)-PLH was as effective as CLC ininhibiting the polymerization of purified tubulin, it was 5-fold lesspotent than CLC in inhibiting the proliferation of A549 cells. Thesedifferences may be due to a low permeability of the cell membrane to(−)-PLH compared to CLC. Alternatively, the difference may be due tointracellular transformation of (−)-PLH to an inactive form. Thecompetitive binding assay using radiolabeled CLC and VLB showed that(−)-PLH inhibited the binding of CLC to tubulin, suggesting that (−)-PLHis a competitive inhibitor of CLC binding to tubulin and its bindingsite may be similar or very close to that of CLC.

[0189] As noted above, several natural and synthetic antimitotic agentshave been reported to inhibit mitosis by binding to the CLC-site ontubulin. See Iwasaki, S., et al., Med. Res. Rev., (1993), 13, 183-98;Hamel, E., Med. Res. Rev., (1996), 16, 207-31. It seems likely thatthese CLC-site ligands such as CLC, steganacin, see Kupchan, S. M., etal., J. Am. Chem. Soc., (1973), 95, 1335-1336, podophyllotoxin, seeSackett, D. L., Pharmacol. Ther., (1993), 59, 163-228 andcombretastatins (Pettit, G. R., et al., J. Med. Chem., (1995), 38,1666-1672, interact at two hydrophobic sites on tubulin with biarylgroups located at appropriate distances and angles, whereas curacin A,see Verdier-Pinard, P., et al., Mol. Pharmacol., (1998), 53, 62-76,probably interacts in a different manner. In the case of (−)-PLH, thespatial arrangement of two aryl groups, the phenyl group and imidazolemoiety, is probably important for binding to tubulin, because theenantiomer (+)-PLH showed little or no effect on mitosis and tubulinpolymerization (data not shown).

[0190] Recently, Usui and colleagues, see Usui, T., et al, Biochem, J.,(1998), 333, 543-48; Kondoh, M., et al., J. Antibiot, (1998), 51,801-04, reported that tryprostatin A and its related compounds, whichare diketopiperazines consisting of isoprenylated tryptophan andproline, affect the microtubule assembly. They also showed thattryprostatin A inhibited microtubule polymerization by interacting withMAP2/tau-binding site rather than with CLC binding site, at relativelyhigher concentrations, see Usui, T., et al., Biochem, J., (1998), 333,543-48; Kondoh, M., et al, J. Antibiot., (1998), 51, 801-04. Althoughtryprostatin A and (−)-PLH have a similar structural motif, thediketopiperazine ring composed of isoprenylated heterocyclic aminoacids, the binding site and effective concentration against microtubulepolymerization are different. It is interesting that the twodiketopiperazine compounds, which have different constituent aminoacids, bind to distinct sites of tubulin molecule and exert a similarbiological activity.

EXAMPLE 12 In Vitro Antitumor Activity of PLH Against Human Cancer CellLines

[0191] For this and the following three examples, EXAMPLES 13, 14 and15, phenylahistin was prepared as described above in EXAMPLE 1.Agar-cultured medium of Aspergillus ustus NSC-F038 was extracted withethyl acetate, and the extract was purified by silica gel columnchromatography twice. PLH was then precipitated in ethyl acetate as awhite powder which contained (−) and (+)-PLH. Two separate batches ofPLH were used, the (−)-PLH contents of which has been analyzed by chiralHPLC. One contained 24.1% (−)-PLH, which was used for evaluating invitro antitumor activity and antitumor activity against P388 Leukemia invivo (Examples 12 and 13), and the other contained 41.7% (−)-PLH, whichwas used for evaluation of in vivo antitumor activity against Lewis LungCarcinoma and Colon 26 (Examples 14 and 15).

[0192] The antitumor activity of PLH was evaluated by the Human CancerCell Line Panel (HCC panel) assay, see Yamori T., Jap. J CancerChemother., (1997), 24, 129-35. This HCC panel consists of 38 tumor celllines including 7 lung cancer, 6 stomach, 6 colon, 5 ovary, 6 centralnerve system, 5 breast, 2 kidney and 1 melanoma cell lines. Each tumorcell was seeded in 96-well tissue culture plates and incubatedovernight. Various concentrations (5 doses; final 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷and 10⁻⁸ M) of PLH were added, and the cells were incubated for 48hours. The number of living cells was then measured using thesulforhodamine B assay, see Keepers Y. P., et al., Eur. J. Cancer, 27,897-900 (1991), and the drug concentration that inhibited cell growth by50% of control cell growth (GI₅₀), the drug concentration that inhibitedcell growth by 100% of control cell growth (TGI) and the drugconcentration that reduced the number of living cells by 50% of theinitial cell number (LC₅₀) then obtained. All mice were obtained fromJapan SLC (Shizuoka, Japan), P388 leukemia cells were obtained fromDainippon Pharmaceuticals Inc. (Osaka, Japan), Lewis Lung Carcinoma fromRiken Cell Bank (Tsukuba, Japan), and Colon 26 cells from the NationalCancer Center, Japan (Tokyo, Japan).

[0193] The results of the HCC panel assay (GI₅₀, TGI, and LC₅₀ for 38cell lines) are shown in Table 5. PLH [(−)-PLH content: 24.1%] exhibitedanti-proliferative activity against 38 cell lines with GI₅₀ valuesranging from 2.3×10⁻⁷M to 4.0×10⁻⁵M. The mean graph, see Yamori T., Jap.J. Cancer Chemother. (1997) 24, 129-135, of GI₅₀ was used for analyzingthe mode of action of newly found antitumor agents. The GI₅₀ mean graphof PLH revealed that the inhibition profile, showing a relatively wideantitumor spectrum, was similar to that of the vinca alkaloids orpaclitaxel, suggesting that the target of the action of PLH may be themicrotubule system. TABLE 5 Cancer Cell Line GL₅₀ (M) TGI (M) LC₅₀ (M)Breast HBC-4 4.6 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ BSY-1 2.3 × 10⁻⁷  6.7 ×10⁻⁷ >1.0 × 10⁻⁴ HBC-5 6.0 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ MCF-7 4.7 ×10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ MDA-MB-231 6.4 × 10⁻⁷  6.0 × 10⁻⁶ >1.0 ×10⁻⁴ CNS U251 4.4 × 10⁻⁷  1.0 × 10⁻⁵ >1.0 × 10⁻⁴ SF-268 4.1 × 10⁻⁷ >1.0× 10⁻⁴ >1.0 × 10⁻⁴ SF-295 3.2 × 10⁻⁷  2.4 × 10⁻⁶ >1.0 × 10⁻⁴ SF-539 2.3× 10⁻⁷  6.0 × 10⁻⁷  1.1 × 10⁻⁵ SNB-75 3.7 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴SNB-78 1.3 × 10⁻⁶ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ Colon HCC2998 1.1 × 10⁻⁶  5.3× 10⁻⁶ >1.0 × 10⁻⁴ KM-12 3.8 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ HT-29 4.0 ×10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ WiDr 2.7 × 10⁻⁷  7.9 × 10⁻⁷ >1.0 × 10⁻⁴HCT-15 4.9 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ HCT-116 4.8 × 10⁻⁷ >1.0 ×10⁻⁴ >1.0 × 10⁻⁴ Lung NCI-H23 9.1 × 10⁻⁷  9.2 × 10⁻⁵ >1.0 × 10⁻⁴NCI-H226 4.1 × 10⁻⁷  3.1 × 10⁻⁵ >1.0 × 10⁻⁴ NCI-H522 2.5 × 10⁻⁷  8.1 ×10⁻⁷ >1.0 × 10⁻⁴ NCI-H460 4.1 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ A549 9.7 ×10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ DMS273 2.9 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴DMS114 3.3 × 10⁻⁷  2.4 × 10⁻⁶ >1.0 × 10⁻⁴ Melanoma LOX-IMVI 1.3 ×10⁻⁶ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ Ovary OVCAR-3  30 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 ×10⁻⁴ OVCAR-4 4.0 × 10⁻⁵ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ OVCAR-5 1.2 × 10⁻⁶ >1.0× 10⁻⁴ >1.0 × 10⁻⁴ OVCAR-8 5.2 × 10⁻⁷  5.6 × 10⁻⁵ >1.0 × 10⁻⁴ SK-OV-33.7 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ Renal RXF-6311 1.9 × 10⁻⁶ >1.0 ×10⁻⁴ >1.0 × 10⁻⁴ ACHN 2.8 × 10⁻⁵ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ Stomach St-44.0 × 10⁻⁵ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ MKN1 4.0 × 10⁻⁷  2.1 × 10⁻⁵ >1.0 ×10⁻⁴ MKN7 3.9 × 10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ MKN28 4.2 × 10⁻⁷ >1.0 ×10⁻⁴ >1.0 × 10⁻⁴ MKN45 1.5 × 10⁻⁶ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴ MKN74 4.8 ×10⁻⁷ >1.0 × 10⁻⁴ >1.0 × 10⁻⁴

[0194] The TGI values were relatively high, i.e., even at 100 μM PLH,and proliferation of 25 of the 38 cell lines was not completelysuppressed. PLH did not exhibit intrinsic cytotoxicity under theseexperimental conditions, and significant LC₅₀ values were not obtainedexcept for SF-539 cells.

EXAMPLE 13 In Vivo Antitumor Activity against P388 Leukemia ofPhenylahistin (PLH)

[0195] Male CDF1 mice (4 weeks old) were inoculated intraperitoneallywith P388 leukemia cells (1×10⁶ cells/body) on day 0. PLH [(−)-PLHcontent; 24%] was suspended in 10% ethanol-saline, and administereddaily at a dose of 4.2 mg/kg, 12.5 mg/kg, 42 mg/kg or 125 mg/kgintraperitoneally, as a single injection (0.2 ml/body), from days 1 to12. Each group consisted of six mice. A T/C (%) was calculated accordingto the following formula:

T/C (%)=[(mean number of survival days of treated group)/(mean number ofsurvival days of control group)]×100

[0196] The antitumor activity of PLH [(−)-PLH content: 24.1%] in miceinoculated intraperitoneally with murine P388 leukemia is shown in Table6. PLH showed significant antitumor activity with a T/C of 129% and 151%at a daily dosage of 41.5 (the calculated dose of (−)-PLH) and 124 (30)mg/kg respectively, for 12 days. The increases in body weight seen inthe control group were also suppressed by daily administrations of PLH41.5 and 124 mg/kg. TABLE 6 Antitumor Activity of PLH against P388Leukemia 4.2 mg/kg 12.5 mg/kg 42 mg/kg 125 mg/kg Control (1 mg/kg)# (3mg/kg)# (10 mg/kg)# (30 mg/kg)# Mean Body weight 25.2 ± 0.6 25.3 ± 0.424.1 ± 0.6 23.0 ± 0.5 22.3 ± 0.3 on day 12 (g) Mean Number of 12.5 ± 0.414.6 ± 0.6* 14.6 ± 0.5** 16.1 ± 0.8** 18.9 ± 0.9** Survival days T/C (%)100 117 117 129 151

[0197] Many fungal diketopiperadine metabolites isolated as micotoxins,for example, fumitremorgin B, a diketopiperazine metabolite consistingof proline and isoprenylated tryptophane and identified as cell cycleinhibitors, exert tremorgenic activity when administered to mice. Inspite of the structural and biological similarities between PLH andfumitremorgin B, PLH did not induce such side effects, even at thehighest dose (124 mg/kg/day). This indicates that the difference ofconstituents in diketopiperazines may be responsible for the distinctpharmacological effect of PLH. Also, PLH seems not to be long-acting invivo, as a single administration of PLH (415 mg/kg PLH [(−)-PLH 100mg/kg]) was not particularly effective (T/C=115%).

EXAMPLE 14 In Vivo Antitumor Activity Against Lewis Lung Carcinoma

[0198] Male BDF1 mice (6 weeks old) were inoculated subcutaneously inthe right flank region with Lewis Lung Carcinoma cells (1×10⁵cells/body) on day 0. PLH [(−)-PLH content; 41.7%] was suspended in 10%ethanol-saline, and was administered daily at a dose of 24 mg/kg, 72mg/kg, or 240 mg/kg intraperitoneally, as a single injection (0.2ml/body), from days 1 to 14. Each group consisted of six mice. Mice wereweighed, and the length (L) and the width (W) of the tumor were measuredthree times a week. On day 15, mice were sacrificed, and the tumor wasexcised and weighed. The estimated tumor weight (ETW) was calculatedaccording to the following formula:

ETW (mg)=L (mm)×W ² (mm² )/2,

[0199] where L represents the length of the tumor and W represents thewidth of the tumor. The suppression rate was calculated according to thefollowing formula:

Suppression rate (%)=[1×(TWt/TWc)]×100,

[0200] where TWt represents the mean tumor weight of the treated groupand TWc indicates that of the control group. Average and standard errorswere calculated for each group. Variance analysis was performed by theBartlett method (significance level: 5%), and if the variance wasuniform, a Dunnett's multiple comparison test was performed. However, ifthe variance was not uniform, rank conversion was performed followed byvariance analysis again by the Bartlett method (significance level: 5%).Significance was established at the p<0.05 level. Based on the weight ofthe excised tumors in the control group, the suppression rate wascalculated for each group, and the ED₅₀ and 95% confidence limit werethen determined by linear regression analysis.

[0201] None of the control and 24 mg/kg/day group mice died. In the 72mg/kg/day group, 1 mouse died on day 11, and in the 240 mg/kg/day group,1 mouse died on day 13.

[0202] In the control group, body weight changed very little during drugadministration. Similarly, little change in body weight was observed inthe 24 and 72 mg/kg/day groups. However, in the 240 mg/kg/day group, thebody weight was markedly lower than control values after day 3, andsignificantly lower on day 6.

[0203] In the control group, the estimated tumor weight (ETW) was 105 mgon day 7, and gradually increased to 2,306 mg on day 15. The ETW wasslightly lower in the 24 mg/kg/day group, in which it increased from 86mg on day 7 to 2,244 mg on day 15. Similarly, the ETW was slightly lowerin the 72 mg/kg/day group, in which it increased from 34 mg on day 7 to2,142 mg on day 15. In the 240 mg/kg/day group, the ETW was the lowest,increasing from 34 mg on day 7 to 617 mg on day 15. Significantsuppression of the ETW was observed on days 10, 13 and 15 (81.8, 82.3and 73.2%, respectively).

[0204] The weight of the excised tumor on the fifteenth day was 3,264 mgfor the control group, and 2,941 and 2,380 mg for 24 and 72 mg/kg/daygroups, respectively, indicating a rate of suppression of 9.9% and27.1%, respectively. In the 240 mg/kg/day group, the excised tumorweight was 619 mg, indicating a rate of suppression of 81.0%. The ED₅₀and 95% confidence limit was 105.3 mg/kg/day [calculated dose of(−)-PLH: 43.9 mg/kg/day] and 22.0˜504.1 mg/kg/day [(−)-PLHL: 9.2˜210.2mg/kg/day], respectively.

EXAMPLE 15 In Vivo Antitumor Activity Against Colon 26

[0205] Male CDFI mice (6 weeks old) were inoculated subcutaneously inthe right flank region with Colon 26 cells (1×10⁵ cells/body) on day 0.Each group consisted of six mice. Preparation and administration of PLH,and the evaluation of antitumor activity was carried out as describedfor Lewis Lung Carcinoma, in EXAMPLE 14.

[0206] The antitumor activity of PLH ((−)-PLH content: 41.7%) in micesubcutaneously inoculated Colon 26 cells was determined. No mice diedduring the experimental period in the control, 24 mg/kg/day and 72mg/kg/day groups. However, in the 240 mg/kg/day group, one mouse died onday 12, two died on day 13 and one died on day 14. No marked changes inthe weight of the control was observed, 24 mg/kg/day and 72 mg/kg/daygroups, whereas, in the 240 mg/kg/day group, the body weight was lowerthan that of the control group from day 3, and was lower from day 3 today 10.

[0207] In the control group, the ETW gradually increased from 51 mg onday 7 to 1067 mg on day 15. In the 24 mg/kg/day and 72 mg/kg/day groups,the ETWs increased but the increments were slightly lower than those ofthe control group. In the 240 mg/kg/day group, the increments of ETWwere significantly lower at 14 mg on day 7 and 254 mg on day 15. Thesuppression rates were 64.4% on day 10, 78.8% on day 13 and 85.4% on day15.

[0208] The weight of the excised tumor on the fifteenth day was 1,159 mgfor the control group, and 1,113 and 843 mg for 24 and 72 mg/kg./daygroups, respectively, indicating a rate of suppression of 4.0% and27.3%, respectively. In the 240 mg/kg/day group, the excised tumorweight was 211 mg, indicating a rate of suppression of 81.8%. The ED₅₀and 95% confidence limit was 107.3 mg/kg/day [calculated dose of(−)-PLH: 44.7 mg/kg/day] and 29.0˜397.7 mg/kg/day [(−)-PLH: 12.1˜165.8mg/kg/day], respectively.

[0209] The in vitro antitumor activity of a scalemic mixture of (−)-PLHand (+)-PLH using HCC panel assay consisting of 38 human cell lines andin vivo antitumor activity using P388 Leukemia, Lewis Lung Carcinoma,and Colon 26 cells were also analyzed in light of the data illustratingthat (−)-PLH exhibits anti-proliferative activity against P388 cellswith an IC₅₀ value of 3.5×10⁻⁷ M and completely inhibits the cell cycleprogression of P388 cells in G2/M phase at 1×10⁻⁶ M, while (+)-PLHexhibited little or no effect on proliferation or cell cycle progressioneven at higher concentrations. See EXAMPLES 3, 4, 5, and 12, 13, 14. Itwas concluded that the antitumor activity of PLH was due to the activeS-configuration enantiomer, (−)-PLH.

EXAMPLE 16 Activity of (+)- and (−)-PHL Against Human Cancer Cell Lines

[0210] In order to examine detailed biological activity, especiallyantitumor activity of both (+)- and (−)-phenylahistin, resolution of(+)- and (−)-phenylahistin using chiral HPLC was conducted. By repeatingchiral separation twice or three times, each enantiomer with an opticalpurity over 99.8% was obtained, and each enantiomer was recrystallizesfrom ethanol-hexane mixture. Antitumor activity of each enanriomer issummarized in Table 7. TABLE 7 Antiproliferative activities of (−)-PLHand (+)-PLH IC₅₀ (M) Cell Line (position) (−)-phenylahistin(+)-phenylahistin A-431 (damal) 2.2 × 10⁻⁷ 2.0 × 10⁻⁵ A549 (lung) 3.0 ×10⁻⁷ 3.0 × 10⁻⁵ Hela (ovary) 2.0 × 10⁻⁷ 1.0 × 10⁻⁵ MCF7 (breast) 3.3 ×10⁻⁷ 1.1 × 10⁻⁵ TE-671 (CNS) 3.7 × 10⁻⁶ 1.3 × 10⁻⁴ WiDr (colon) 1.8 ×10⁻⁷ 8.5 × 10⁻⁶ K562 (leukemia) 1.9 × 10⁻⁷ 1.0 × 10⁻⁵

[0211] Antitumor activity of (+)- and (−)-phenylahistin was examinedagainst seven human cancer cell lines including A431, WiDr, TE-671,Hela, MCF7, A-549 and K562 cells. The IC₅₀ values of each enantiomerwere shown in Table 7. (−)-Phenylahistin exhibited antitumor activitywith the IC₅₀ values ranging from 1.8×10⁻⁷ (against WiDr cells) to3.7×10⁻⁵ (against TE-671). While (+)-PLH exhibited approximately 33 to100-fold less activity than (−)-PLH.

EXAMPLE 17 Syntheses and Biological Activity of PhenylahistinDerivatives: Criterion to Distinguish Biologically Active Derivativesfrom Relatively Less-Active Derivatives

[0212] To characterize the anti-tumor phenylahistin derivatives of thepresent invention, the structural factors that are required to exhibitthe anti-microtubule activity of these compounds have been determined.These factors serve to distinguish active derivatives of the inventionfrom relatively less-active compounds.

[0213] In this and the following example, X-ray crystallographicanalyses of phenylahistin, the synthesis and physical characterizationof various phenylahistin derivatives, and analyses of the structuralfactors necessary for anti-microtubule activity were conducted. Theseanalyses, which were focused on the structure of unique isoprenylateddehydrohistidine, indicate that this isoprenylated dehydrohistidineforms a rigid planar pseudo three-ring structure, composed of thediketopiperazine and imidazole rings through a hydrogen bond betweenN8—H and N3 and α,β-unsaturated bond (C6—C7). It is concluded that thisstructural feature is important for the activity of the anti-tumoractivity of the compounds of the present invention.

[0214] The derivatives analyzed in this and the following example wereprepared in the following manner. As shown in FIG. 3 (Reaction Scheme3), simple alkylation and reduction of phenylahistin (labeled “11” inFIG. 3) and the modification of cyclo(Gly-Phe) with imidazolederivatives were useful to synthesized the derivatives of phenylahistinfor analyzing the structural factors. Derivatives 12-15 were synthesizedfrom phenylahistin by alkylation or reduction. As shown in FIG. 3,Reaction Scheme 2, compounds 12 and 13 were synthesized by hydrogenationof each enantiomer of phenylahistin over 10% palladium on carbon in MeOHunder atmospheric hydrogen at room temperature. Two (2) hours ofhydrogenation yielded derivative 12, in which the1,1-dimethyl-2-propenyl group of phenylahistin was reduced, and furtherhydrogenation (24 h) of compound 12 yielded compound 13, in which thedehydrohistidine part was reduced. In this second reduction, only onediastereomer, which has the same polarity in optical rotation as that ofcompound 12, was obtained. The diastereoselective hydrogenation ofcompound 13 may be due to the steric hindrance of the phenyl ring of thePhe residue.

[0215] As shown in FIG. 4, Reaction Scheme 4, methylation ofphenylahistin (labeled “1” in FIG. 4) with MeI and NaH in DMF gave mono-and tri-methylated compounds. The extent of the methylation could becontrolled by reaction temperature rather than amounts of the reagents.Mono-methylated derivative 14 at the τ nitrogen of the imidazole ring,whose methylation site was detected by NMR analysis (note that the lowfiled shift of 8(N)—H (δ 12.20 ppm) in the ¹H NMR spectrum wasmaintained in compound 14) and was predominantly obtained withequivalent of the reagents at −30° C., and a tri-methylated derivative15 was obtained in the reaction with 30 equivalents of the reagents atroom temperature. Compound 14 and 15 with the L-phenylalanine residuewere separated by HPLC with a chiral column.

[0216] As shown in FIG. 5, Reaction Scheme 5, compounds 19 and 20 weresynthesized from cyclo(Gly-Phe), compound 16, that was prepared bycyclization of H-Phe-Gly-OMe. After acetylation of the two amidenitrogens of the diketopiperazine ring in 16,4(5)-imidazolecarboxaldehyde or 4-methyl-5-imidazolecarboxaldehyde (18)was introduced in the presence of lithium diisopropylamide (LDA) andhexamethylphosphoramide (HMPA), according to the method of Bond, R., etal. Synthetic Commun., 19, 2551 (1989), and subsequent dehydration withtriflic anhydride-pyridine and deacetylation with aqueous NH₄OH gavecompounds 19 and 20, although less than 20% of racemization was observedin these steps.

[0217] According to X-ray crystallographic analyses of(+)-phenylahistin, as provided in Table 8, the stereochemistry of C6—C7double bond was confirmed to be Z, and the existence of a hydrogen bondbetween N8—H and N3 was observed. This result was in good agreement withthe observation in the NMR studies (low field-shift of N8—H (δ 12.08ppm). These findings suggested that two heterocycles, i.e., thediketopiperazine and imidazole rings, were fixed in the same plane byforming a pseudo three-ring structure. The benzyl group of the Pheresidue was stacked over the diketopiperazine ring by sticking out fromthis plane, whose conformation is reported as the energetically mostfavored one in diketopiperazine with an aromatic amino acid residue,according to the method of Liwo, A. et al. Tetrahedron Lett., 26, 1873(1985). The 1,1-dimethyl-2-propenyl group at the imidazole ring was alsorestricted its movement by the steric hindrance of a hydrogen atom atthe β-position (C6) of the α,β-unsaturated His derivative. Therefore, itis indicated that the conformation of (+)-phenylahistin is highlyrestricted. Since the (−)-form of 11, i.e., the biologically activeenantiomer, also takes the same conformational feature as that of its(+)-form only with the opposite configuration at the α-position of thePhe residue, this rigid conformation of phenylahistin may be importantfor the binding to the microtubule protein. TABLE 8 Data of the x-raycrystallographic analysis of (+)-phenylahistin Crystal parameters —Empirical formula C₂₀H₂₂N₄O₂ Formula weight  350.42 Crystal systemtetragonal Lattice parameters: a = 15.3509(7) Å c = 8.309(2) Å V =1958.0(2) Å³ Z = 4 Space group P4₂ (#77) Dcalc. 1.189 g/cm³ μ (CuKα)6.37 cm⁻¹ Refinement parameters — Reflections measured 2229 Nonzeroreflections 1724 R-indexResiduals: R^(a)   0.057 Residuals: RW^(b)  0.067 Goodness of fit indicator^(c)   4.31

[0218] To study the relationship between the rigid plane structure of(−)-phenylahistin and that compound's biological activity, thederivatives of (−)-phenylahistin were synthesized and theanti-microtubule effect on the polymerization of microtubule proteinprepared from bovine brain and the anti-proliferative effect on P388cells were studied, as described in prior examples. As described above,(−)-phenylahistin exhibits the colchicine-like inhibition of microtubulepolymerization (IC₅₀=25 μM). This inhibitory activity of(−)-phenylahistin was almost the same as that of colchicine (IC₅₀=16μM), although the anti-proliferative activity of (−)-phenylahistin(IC₅₀=0.21 μM) was about 10 times less active than that of colchicine(IC₅₀=0.031 μM). The difference of these activities is due either to theunaccounted-for biological activities of colchicine or to lower cellularpermeability of (−)-phenylahistin in anti-proliferative assay, since(−)-phenylahistin has more hydrophilic structure than colchicine.Furthermore, as described above, (−)-phenylahistin with the L-Pheresidue was 50 times more active than (+)-phenylahistin inanti-proliferative assays. (+)-Phenylahistin also exhibited relativelyweak inhibitory activity against microtubule polymerization (15%inhibition at 200 μM) compared with (−)-phenylahistin. Thus, theorientation of the benzyl group of the phenylalanine residue isimportant to the biological activity of the compounds of the presentinvention.

[0219] As shown in Table 9, compound 12, in which the1,1-dimethyl-2-propenyl group of phenylahistin was reduced, showed thesame activity as phenylahistin, indicating that this double bond is notimportant for the anti-microtubule activity. However, compound (−)-13,in which the dehydrohistidine of (−)-12 was reduced, completely lost theinhibitory activity. Since this modification disrupts the substantiallyplanar characteristic of the pseudo three-ring structure in the sameplane as the phenylahistin backbone, this result suggests that, for theinhibitory activity, it is important that the two rings, i.e.,diketopiperazine and imidazole rings, are fixed in the same plane. TABLE9 Biological activity of phenylahistin, its derivatives, and selectedstructurally-related compounds. IC₅₀ (μM) Microtubule Protein^(a) P388compound polymerization proliferation (−)-11  25  0.21 (±0.02)^(b)(+)-11 >200 [15%]^(c)   10 (±1.5) (−)-12  30  0.23 (±0.05) (+)-12 >200[12%]   19 (±4.2) (−)-13 >200 >200 (+)-13 >200 >200 14  100  0.95(±0.03) 15 >200   160 (±5.5) 19 >200 [11%] >200 20 >200 {9%]   15 (±4.5)Cyclo(His-Phe)^(d) >200 >200 colchicine^(d)  16 0.031 (±0.01)

[0220] In further syntheses, the nitrogen atoms of phenylahistin weremodified by methylation. Compound 14, in which the τ nitrogen of theimidazole ring of (−)-phenylahistin was methylated, was about 5 timesless active than (−)-phenylahistin, suggesting this imidazole nitrogenprobably participates in the binding with the microtubule protein orthis methylation affords the structural hindrance to the conformation ofthe 1,1-dimethyl-2-propenyl group on the imidazole ring. Tri-methylatedcompound 15 almost lost the inhibitory activity. This result alsosuggests that the rigid plane conformation of (−)-phenylahistin isimportant to the anti-microtubule activity, since the methylation of adiketopiperazine nitrogen (N8) disrupts the formation of hydrogen bondon N8—H necessary for the rigid pseudo three-ring conformation.

[0221] The importance of an alkyl group at the 5-position on theimidazole ring to the biological activity of the compounds of thepresent invention was analyzed via the following analyses of compounds19 and 20, which were also synthesized from cyclo(Phe-Gly), compound 16.In compounds 19 and 20, the 1,1-dimethyl-2-propenyl group of(−)-phenylahistin was replaced with a hydrogen atom or a methyl group,respectively. Compound 19, in which the 1,1-dimethyl-2-propenyl groupwas replaced with the hydrogen atom at this position, showed noinhibitory activity. Compound 20, in which the 1,1-dimethyl-2-propenylgroup was replaced with a methyl group at this position, also showed noactivity in the anti-microtubule assay, although weak inhibition on P388cell proliferation was observed. This drastic decreased in biologicalactivity in 19 indicates that an alkyl group with a proper length or aquaternary carbon at the 5-position of the imidazole ring is importantfor the activity.

[0222] Accordingly, the results of the x-ray crystallographic analysisand the biological evaluation of the phenylahistin derivatives elucidatea structural factor of phenylahistin necessary for its anti-microtubuleactivity. This factor is that the rigid and planar pseudo three-ringstructure formed by a hydrogen bond are important for the biologicalactivity of (−)-phenylahistin.

[0223] For this example, the microtubule protein polymerization assaywas performed as follows. Microtubule protein was prepared from bovinebrain tissue by two cycles of assembly and disassembly. Polymerizationof microtubule protein was monitored by an increase in turbidity at 37°C. in microtubule assembly buffer containing 100 mM MES, 0.5 mM MgCl₂, 1mM EGTA and 1 mM GTP. The polymerization of microtubule protein wasinitiated by a temperature shift from 0° C. to 37° C. Turbidity wasmeasured on a thermo-controlled spectrophotometer (Beckman DU-20,Fullerton, Calif.) at 360 nm. Compounds were dissolved in DMSO, whichwas used in all experiments at a final concentration of 2% (v/v).

[0224] For this example, the Alamar Blue™ assay was performed asfollows. Exponentially growing P388 cells were seeded into 96-welltissue culture plates (5×10³ cells/100 μl/well) and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum for 16 h. Compounds(DMSO solution) were then added to each well at various concentrations,and the cells were cultured for an additional 48 h. Living cells werecounted using Alamar Blue™ (BioSource International, Camarillo, Calif.),according to the method of Ahmed, S. A. et al., J. Immunol. Methods,170, 211 (1994).

[0225] For this example, the Single-Crystal X-ray Diffraction Analysiswas performed as follows. A pale yellow crystal of (+)-phenylahistinhaving approximately dimension 0.4×0.4×0.3 mm, which was crystallizedfrom EtOAc, was used for single-crystal x-ray diffraction analysis. Allx-ray measurements were made on a Rigaku AFC7R diffractometer withgraphite monochromated Cukα radiation and a rotating anode generator.The crystal data and refinement parameters were summarized in Table 8.Of the 2229 reflections which were collected, 2064 were unique(Rint=0.019). The data were corrected for Lorentz and polarizationeffects. A correction for secondary extinction was applied. Thestructure was solved by direct methods and expanded using standardFourier techniques. All calculations were performed using the teXscancrystallographic software package of Molecular Structure Corporation.

EXAMPLE 18 Syntheses and Physical Characterization of PhenylahistinDerivatives

[0226] The melting points of the phenylahistin derivatives described inthe previous example were determined on a Mettler FP62 apparatus and areuncorrected. ¹H-NMR and 13C-NMR spectra were recorded on JEOL GSX270Jspectrometer. The spectra were recorded with tetramethylsilane (δ=0.0for ¹H); DMSO-d₆ (δ=39.5 for ¹³C); CDCl₃ (δ=77.0 for ¹³C) as internalreference. Mass spectra (electrospray ionization, methanol as the mobilephase) were analyzed with Finnigan SSQ 7000 spectrometer.High-resolution fast atom bombardment mass spectra were analyzed withJEOL JMS-DX303 spectrometer. Infrared spectra were recorded on a JEOLJIR-5500 infrared spectrophotometer in KBr pellets. A silica-gel columnchromatography was performed using Merck 70-230 mesh silica gel 60.Optical rotations were measured on a Horiba SEPA-200 polarimeter, andare given in units of 10⁻¹ deg cm² g⁻¹. Elemental analyses wereperformed by Fisons EA 1108 elemental analyzer.

[0227] The precise synthetic routes and physical characteristics of thephenylahistin derivatives described in the previous example are asfollows.

[0228](−)-Cyclo-[5-(1,1-dimethylpropyl)dehydrohistidinyl-L-phenylalanine](—)-12:

[0229] To a solution of (−)-phenylahistin (30 mg, 0.086 mmol) in MeOH(10 mL) was added 10 mg of 10% palladium on carbon and the mixture wasstirred at room temperature for 2 h under hydrogen at atmosphericpressure. After the catalyst was filtered off and the filtrate wasconcentrated under reduced pressure, the residue was purified by columnchromatography on silica (5 g) using CHCl₃-MeOH (50:1) as an eluent.Desired fractions were collected and the solvent was evaporated. Theresidual white powder was recrystallized from EtOH-hexane to give 12 mg(39% of (−)-12 as a colorless solid: mp 224-226° C.; [α]_(D) ²⁵−295(c=0.15, MeOH), UV (MeOH) nm 322 (ε 23300), 231 (ε 8830), 203 (ε 16800);IR (KBr) cm⁻¹ 3310, 3220, 2970, 1670, 1440; ¹H NMR (270 MHz, CDCl₃) δ12.09 (br s, 1H), 8.98 (br s. 1H), 7.56 (s, 1H), 7.29-7.38 (m, 5H), 6.87(s, 1H), 5.67 (s, 1H), 4.34 (ddd, J=2, 3, 10 Hz, 1H), 3.51 (dd, J=3, 14Hz, 1H), 2.29 (dd, J=10, 14 Hz, 1H), 1.74 (q, J=7 Hz, 2H), 1.40 (s, 6H),0.74 (t, J=7 Hz 3H); ¹³C NMR (67.5 MHz, CDCl₃) δ 164.6, 159.9, 138.1,135.5, 132.2, 132.1, 129.5 (2C), 129.1 (2C), 127.4, 123.6, 105.4, 57.2,41.3, 36.2, 35.4, 27.9 (2C), 9.2; HRMS m/z 352.1926 (M⁺) (calcd forC₂₀H₂₄N₄O₂: 352.1899). Anal. calcd for C₂₀H₂₄N₄O₂: C, 68.16; H, 6.86; N,15.90. Found: C, 68.34, 6.78, 15.82.

[0230](+)-Cyclo-[5-(1,1-dimethylpropyl)dehydrohistidinyl-L-phenylalanine](+)-12: This compound was prepared according to the same procedure forthe preparation of (−)-12. 44% yield from (+)-1; colorless solid; mp222-223° C.; [α]_(D) ²⁵+284(c=0.10, MeOH), UV (MeOH) nm 322 (ε 22800),231 (ε 8720), 203 (ε 16300); IR (KBr) cm⁻¹ 3310, 3220, 2970, 1670, 1440;¹H NMR (270 MHz, CDCl₃) δ 12.09 (br s, 1H), 8.98 (br s. 1H), 7.56 (s,1H), 7.29-7.38 (m, 5H), 6.87 (s, 1H), 5.67 (s, 1H), 4.34 (ddd, J=2, 3,10 Hz, 1H), 3.51 (dd, J=3, 14 Hz, 1H), 2.29 (dd, J=10, 14 Hz, 1H), 1.74(q, J=7 Hz, 2H), 1.40 (s, 6H), 0.74 (t, J=7 Hz 3H); ¹³C NMR (67.5 MHz,CDCl₃) δ 164.6, 159.9, 138.1, 135.5, 132.2, 132.1, 129.5 (2C), 129.1(2C), 127.4, 123.6, 105.4, 57.2, 41.3, 36.2, 35.4, 27.9 (2C), 9.2; HRMSm/z 352.1932 (M⁺) (calcd for C₂₀H₂₄N₄O ₂: 352.1899). Anal. calcd forC₂₀H₂₄N₄O₂: C, 68.16; H, 6.86; N, 15.90. Found: C, 68.09, 6.87, 15.84.

[0231] (−)-Cyclo-[5-(1,1-dimethylpropyl)histidinyl-L-phenylalanine](−)-13: This compound was prepared from (−)-12, according to the sameprocedure for the preparation of (−)-12, with 24 h of reaction time. 60%yield from (−)-12; white powder; mp 224-225° C.; [α]_(D) ²⁵−96(c=0.16,MeOH), UV (MeOH) nm 257 (ε 194), 205 (ε 18100), IR (KBr) cm⁻¹ 3380,3200, 2970, 1670, 1440; ¹H NMR (270 MHz, DMSO-d6) δ 11.55 (br s, 1H),8.25 (br s. 1H), 7.86 (br s, 1H), 741 (s, 1H), 7.29-7.14 (m, 5H), 4.24(br s, 1H), 3.94 (br d, J=11 Hz, 1H), 3.11 (dd, J=14, 4 Hz, 1H), 2.88(dd, J=14,5 Hz, 1H), 2.82 (dd, J=15, 2 Hz, 1H), 1.45 (q, J=7 Hz, 2H),1.32 (dd, J=15, 11 Hz, 1H), 1.13 (s, 3H), 1.12 (s, 3H), 0.60 (t, J=7 Hz3H); ¹³C NMR (67.5 MHz, CDCl₃) δ 166.87, 165.42, 135.93, 132.54, 131.58,131.23, 130.32 (2C), 127.94 (2C), 126.61, 55.70, 54.31, 38.16, 35.04,34.31, 31.85, 27.63, 27.57, 9.05; HRMS m/z 354.2098 (M⁺) (calcd forC₂₀H₂₄N₄O₂: 354.2055). Anal. calcd for C₂₀H₂₄N₄O₂: ⅓H₂O: C, 66.64; H,7.46; N, 15.54. Found: C, 66.75, 7.41, 15.52.

[0232] (+)-Cyclo-[5-(1,1-dimethylpropyl)histidinyl-L-phenylalanine](+)-13:

[0233] This compound was prepared from (+)-12 according to the sameprocedure for the preparation of (−)-13. 46% yield from (+)-12; whitepowder; mp 226-227° C.; [α]_(D) ²⁵+99(c=0.10, MeOH), UV (MeOH) nm 257 (ε174), 205 (ε 18200), IR (KBr) cm⁻¹ 3380, 3200, 2970, 1670, 1440; ¹H NMR(270 MHz, DMSO-d₆) δ 11.55 (br s, 1H), 8.25 (br s. 1H), 7.86 (br s, 1H),7.41 (s, 1H), 7.29-7.14 (m, 5H), 4.24 (br s, 1H), 3.94 (br d, J=11 Hz,1H), 3.11 (dd, J=14, 4 Hz, 1H), 2.88 (dd, J=14,5 Hz, 1H), 2.82 (dd,J=15, 2 Hz, 1H), 1.45 (q, J=7 Hz, 2H), 1.32 (dd, J=15, 11 Hz, 1H), 1.13(s, 3H), 1.12 (s, 3H), 0.60 (t, J=7 Hz 3H); ¹³C NMR (67.5 MHz, CDCl₃) δ166.87, 165.42, 135.93, 132.54, 131.58, 131.23, 130.32 (2C), 127.94(2C), 126.61, 55.70, 54.31, 38.16, 35.04, 34.31, 31.85, 27.63, 27.57,9.05; HRMS m/z 354.2110 (M⁺) (calcd for C₂₀H₂₄N₄O₂: 354.2055). Anal.calcd for C₂₀H₂₄N₄O₂: ½H₂O: C, 66.09; H, 7.49; N, 15.41. Found: C,65.80, 7.50, 15.30.

[0234](−)-Cyclo-[(N^(im)-methyl-5-(1,-dimethyl-2-propenyl))dehydrohistidinyl-L-phenylalanine](−)-14:

[0235] To a solution of phenylahistin (200 mg, 0.57 mmol) in DMF (15 mL)was added 45 mg (1.88 mmol) of sodium hydride (NaH) (60% in mineral oil)in portions and the mixture was stirred at −30° C. for 10 min. To thismixture, 1.0 mL (17.1 mmol) of MeI was added dropwise and stirred at−30° C. for 2 h. 20 mL of saturated aqueous NH₄Cl was added to thereaction mixture and the mixture was extracted three times with 50 mL ofEtOAc. The combined organic layers were washed with saturated NaCl,dried over Na₂SO₄ and concentrated in vacuo. The residual white powder(150 mg) was chromatographed over 20 g of silica gel and eluted withCHCl₃-MeOH (50:1) as an eluent to give 130 mg (63%) of 14 as whitepowder: The (−)-form of 14 was isolated by HPLC using chiral column(CHIRALCEL OD, 10×250 mm) eluted with a mixture of hexane and ethanol(3:1) at a flow rate of 6.0 mL/min on a Waters system (600E series). 19%yield from 1; white powder; mp 214-215° C.; [α]_(D) ²⁵−285(c=0.30,MeOH), UV (MeOH) nm 317 (ε 25400), 232 (sh, ε 8640), 204 (ε 16800); IR(KBr) cm⁻¹ 3200, 2980, 1680, 1440; ¹H NMR (270 MHz, CDCl₃) δ 12.20 (brs, 1H), 7.39 (s. 1H), 7.38-7.24 (m, 5H), 7.09 (s, 1H), 6.01 (dd, J=18, 9Hz, 1H), 5.68 (br s, 1H), 5.14 (d, J=9 Hz, 1H), 5.00 (d, J=18 Hz, 1H),4.33 (m, 1H), 3.66 (s. 3H), 3.50 (dd, J=14, 3 Hz, 1H), 2.92 (dd, J=14,10 Hz, 1H), 1.59 (s, 6H); ¹³C NMR (67.5 MHz, CDCl₃) δ 164.6, 160.1,145.6, 138.5, 136.5, 135.6, 134.6, 129.5 (2C), 129.1 (2C), 127.4, 124.0,112.7, 106.6, 57.1, 41.3, 39.2, 34.9, 28.9, 28.8; HRMS m/z 364.1909 (M⁺)(calcd for C₂₁H₂₄N₄O₂: 364.1899). Anal. calcd for C₂₁H₂₄N₄O₂.¼H₂O: C,68.36; H, 6.69; N, 15.19. Found: C, 68.44, H, 6.67, N, 15.01.

[0236] (−)-Cyclo-N,N′-dimethyl-[(N^(im)-methyl-5-(1,1-dimethyl-2-propenyl))dehydro-histidinyl-L-phenylalanine](−)-15:

[0237] This compound was prepared from phenylahistin with 10 equivalentsof NaH and 30 equivalent of MeI at room temperature according to thesame procedure for the preparation of (−)-14. For the purification of(−)-15, a mixture of hexane and ethanol (4:1) was used as an eluent inthe chiral column HPLC mentioned in the preparation of (−)-14. 8% yieldfrom phenylahistin: white powder; mp 95-98° C.; [α]_(D) ²⁵−632(c=0.50,MeOH), UV (MeOH) nm 287 (ε 11100), 203 (ε 15900); IR (KBr) cm⁻¹ 2900,1690, 1640, 1380; ¹H NMR (270 MHz, CDCl₃) δ 8.76 (br s, 1H), 7.33-7.21(m, 5H), 7.08 (s, 1H), 6.00 (dd, J=18, 11 Hz, 1H), 5.28 (d, J=11 Hz,1H), 5.08 (d, J=18 Hz, 1H), 4.19 (dd, J=10,4 Hz, 1H), 3.86 (s, 3H), 3.38(dd, J=14, 4 Hz, 1H), 3.13 (dd, J=14, 10 Hz, 1H), 2.92 (s, 3H), 2.55 (s,3H), 1.60 (s, 3H), 1.57 (s, 3H); ¹³C NMR (67.5 MHz, CDCl₃) δ 166.8,160.2, 143.5, 137.8, 136.5, 134.8, 129.4 (2C), 128.8 (2C), 127.2, 124.6,114.4, 105.6, 65.9, 40.6, 38.8, 36.6, 34.9, 28.4, 28.1; HRMS m/z392.2223 (M⁺) (calcd for C₂₃H₂₈N₄O₂: 392.2212). Anal. calcd forC₂₃H₂₈N₄O₂: C, 70.38; H, 7.19; N, 14.27. Found: C, 70.26, H, 7.30, N,14.27.

[0238] Cyclo-[glycinyl-L-phenylalanine] 16:

[0239] To a solution of H-Phe-Gly-OMe, which was prepared fromBoc-Phe-Gly-OMe with 4N HCl-dioxane (20 g, 59 mmol), in MeOH (100 mL)was refluxed for 16 h. The white precipitate occurred during reflux waswashed with 10 mL of MeOH for three times and collected to give 7.5 g(62%) of 16 as a white powder, mp 262-263° C. (decomp.); [α]_(D)25+60(c=0.15, DMSO), UV (MeOH) nm 257 (ε 101), 206 (ε 5770); IR (KBr)cm⁻¹3340, 3200, 3060, 1680, 1470, 1340; ¹H NMR (270 MHz, DMSO-d₆) δ 8.16(br s, 1H), 7.90 (br s. 1H), 7.32-7.15 (m, 5H), 4.07 (br dd, J=7,4 Hz,1H), 3.35 (dd, J=18, 3 Hz, 1H), 3.10 (dd, J=14, 4 Hz, 1H), 2.88 (dd,J=14, 5 Hz, 1H), 2.75 (d, J=18 Hz, 1H), ¹³C NMR (67.5 MHz, DMSO-d₆) δ167.1, 165.5, 136.0, 130.1 (2C), 128.1 (2C), 126.8, 55.5, 43.7, 38.8; MS(ESI) m/z 205 (M+H)⁺; Anal. calcd for C₁₁H₁₂N₂O₂. ⅕H₂O: C, 63.57; H,6.01; N, 13.48. Found: C, 63.85, H, 5.86, N, 13.40.

[0240] Cyclo-N,N′-diacetyl-[glycinyl-L-phenylalanine] 17:

[0241] The mixture of compound 16 (0.5 g, 2.45 mmol) and fused sodiumacetate (201 mg, 2.45 mmol) in acetic anhydride (10 mL) was heated for16 h at 100° C. under nitrogen. After acetic anhydride was removed invacuo at 45° C., the residue was solved in EtOAc and the resultedorganic layer was washed with saturated NaCl, dried over Na₂SO₄ and 17was crystallized from EtOAc. 88% yield (0.62 g); colorless solid; mp84-85° C.; [α]_(D) ²⁵+7.8 (c=0.52, MeOH); UV (MeOH) nm 209 (ε 20400); IR(KBr) cm⁻¹ 1720, 1400, 1380, 1240; ¹H NMR (270 MHz, CDCl₃) δ 7.33-7.26(m, 3H), 7.08-7.05 (m, 2H), 5.44 (t, J=5 Hz, 1H), 4.49 (d, J=19 Hz, 1H),3.35 (dd, J=14, 5 Hz, 1H), 3.20 (dd, J=14, 5 Hz, 1H), 2.58 (s, 3H), 2.55(s, 3H), 2.48 (d, J=19 Hz, 1H); ¹³C NMR (67.5 MHz, CDCl₃) δ 171.2,171.0, 167.9, 166.0, 134.3, 129.7 (2C), 129.1 (2C), 128.2, 59.0, 46.0,38.7, 27.1, 26.8; MS (ESI) m/z 311 (M+Na)⁺; Anal. calcd for C₁₅H₁₅N₂O₄:C, 62.49; H, 5.59; N, 9.72. Found: C, 62.50, H, 5.50, N, 9.67.

[0242] Cyclo-[dehydrohistidinyl-L-phenylalanine] 19:

[0243] To a solution of 17 (311 mg, 0.93 mmol) in dimethoxyethanol (DME)(10 mL) was added 0.6 mL of 2M lithium diisopropylamide (LDA, 1.2 mmol)and the mixture was stirred at −70° C. for 10 min. To this solution wasadded a solution of 4(5)-formylimidazole (120 mg, 1.25 mmol, MaybridgeChemical Co. Ltd., Cornwall, U.K.) in HMPA (4 mL)-DME (6 mL) at −70° C.This mixed solution was allowed to warm to −30° C. After stirring for 30min at this temperature, a triflic anhydride (370 μL, 2.2 mmol) andpyridine (180 μL, 2.2 mmol) were added and the solution was allowed towarm to room temperature. After additional stirring for 1 h, aqueousammonia (3 mL) was added and stirred for 14 h at room temperature. Thereaction mixture was extracted with CHCl₃ for three times, dried overNa₂SO₄ and concentrated in vacuo. The resultant residue was purified byHPLC with a reverse phase column (Waters, μBondasphere 19×150 mm, 10 μm,C-18) employing a gradient from 60 to 80% CH₃CN in 0.1% TFA at a flowrate of 17 mL/min on a Waters system (600E series). However, sincepurified compound 19 contained 28% of a racemized compound withD-phenylalanine (44% ee), further purification by HPLC with a chiralcolumn was performed using the same procedure described for thepurification of (−)14. 8% yield from 7; white powder; mp 208-209° C.[α]_(D) ²⁵−257 (c=0.21, DMSO); UV (MeOH) nm 307 (ε 16700); IR (KBr) cm⁻¹3400, 3120, 1680, 1440; ¹H NMR (270 MHz, DMSO-d₆) δ 12.88 (br s, 1H),11.09 (br s, 1H), 8.44 (s, 1H), 8.13 (br s, 1H), 7.48 (s, 1H), 7.24-7.11(m, 5H), 6.25 (s, 1H), 4.48 (m, 1H), 3.19 (dd, J=14, 4 Hz, 1H), 2.95(dd, J=14, 5 Hz, 1H); ¹³C NMR (67.5 MHz, DMSO-d₆) δ 164.8, 158.6, 135.8,135.5, 132.3, 130.0 (2C), 128.0 (2C), 126.7, 125.6, 118.3, 101.8, 55.9,38.9; (overlapping DMSO-d₆); HRMS m/z 282.1084 (M⁺) (calcd forC₁₅H₁₄N₄O₂: 282.1117). Anal. calcd for C₁₅H₁₄N₄O₂—CF₃COOH: C, 51.52; H,3.81; N, 14.14, Found: C, 51.15, H, 3.62, N, 13.84.

[0244] Cyclo-[(5-methyl)dehydrohistidinyl-L-phenylalanine] 20:

[0245] This compound was prepared from compound 17 with4-methyl-5-imidazolecarboxaldehyde according to the same procedure forthe preparation of 19. 3% yield from 17; colorless solid; mp 285-286° C.(decomp); [α]_(D) ²⁵−267° (c=0.21, DMSO); UV (MeOH) nm 319 (ε 22800); IR(KBr) cm⁻¹ 3400, 3180, 1680, 1450; ¹H NMR (270 MHz, DMSO-d₆) δ 11.50 (brs, 1H), 8.35 (br s, 1H), 7.74 (s, 1H), 7.24-7.14 (m, 5H), 6.20 (s, 1H),4.48 (m, 1H), 3.33 (br s, 1H), 3.20 (dd, J=14, 4 Hz, 1H), 2.93 (dd,J=14, 5 Hz, 1H), 2.19 (s, 1H); ¹³C NMR (67.5 MHz, DMSO-d₆) δ 164.2,158.7, 135.6, 134.6, 132.3, 130.0 (2C), 128.0 (2C), 127.5, 126.6, 123.4,101.7, 55.9, 38.7, 8.9; High-resolution MS m/z 296.1261 (M⁺) (calcd forC₁₆H₁₆N₄O₂: 296.1273). Anal. calcd for C₁₆H₁₆N₄O₂ ⅕H₂O; C, 64.07; H,5.51; N, 18.68. Found: C, 64.39, H, 5.65, N, 18.29.

EXAMPLE 19 Exemplary Formulation Administered Intravenously, by Drip,Injection, or the Like

[0246] Vials containing 5 g of powdered glucose are each addedaseptically with 10 mg of the invention compound and sealed. After beingcharged with nitrogen, helium or other inert gas, the vials are storedin a cool, dark place. Before use, the contents are dissolved in ethanoland added to 100 ml of a 0.85% physiological salt water solution. Theresultant solution is administered as a method of inhibiting the growthof a cancerous tumor in a human diagnosed as having such a tumor atbetween approximately 10 ml/day to approximately 1000 ml/day,intravenously, by drip, or via a subcutaneous or intraperitonealinjection, as deemed appropriate by those of ordinary skill in the art.

EXAMPLE 20 Exemplary Formulation to be Administered Orally

[0247] A mixture obtained by thoroughly blending 1 g of the compound ofthe invention, 98 g of lactose and 1 g of hydroxypropyl cellulose isformed into granules by any conventional method. The granules arethoroughly dried and sifted to obtain a granule preparation suitable forpackaging in bottles or by heat sealing. The resultant granulepreparations are orally administered at between approximately 100 ml/dayto approximately 1000 ml/day, depending on the symptoms, as deemedappropriate by those of ordinary skill in the art of treating canceroustumors in humans.

EXAMPLE 21 Use of Phenylahistin to Treat a Human Suffering from Cancer

[0248] PLH is administered according to the formulation of EXAMPLE 17 toa human patient afflicted with lung cancer. PLH is administered atbetween approximately 10 mg per day to approximately 1000 mg per day,and in an preferred amount of 50 mg per day, until the growth of thetumor is inhibited.

EXAMPLE 22 Use of (−)-Phenylahistin to Treat Human Suffering from Cancer

[0249] (−)-PLH is isolated according to the method of EXAMPLE 6,substantially purified, and administered according to the formulation ofEXAMPLE 17 to a human patient, afflicted with ovarian cancer, at betweenapproximately 10 mg per day to approximately 100 mg per day, and in apreferred amount of 50 mg per day, until the growth of the tumor isinhibited.

EXAMPLE 23 Anti-Tumor Effect of 2-Isopropyl-(−)-Phenylahistin

[0250] 2-isopropyl-(−)-phenylahistin, i.e., compound 3 of EXAMPLE 7, issynthesized according to the method of EXAMPLE 7, substantiallypurified, and administered according to the formulation of EXAMPLE 17 toa human patient afflicted with breast cancer at between approximately 10mg per day to approximately 100 mg per day, and in a preferred amount of50 mg per day, until the growth of the tumor is inhibited.

EXAMPLE 24 Synthesis of 2-Dichloroisopropyl-(−)-Phenylahistin

[0251] 2-dichloroisopropyl-(−)-phenylahistin is synthesized in thefollowing manner: (−)-Phenylahistin, compound 1 of EXAMPLE 7, is placedin the presence of Cl₂ and in the presence of water and heat to yield 2dichloroisopropyl-(−)-phenylahistin.

EXAMPLE 25 Anti-Tumor Effect of 2-Dichloroisopropyl-(−)-Phenylahistin

[0252] 2-dichloroisopropyl-(−)-phenylahistin, as synthesized in EXAMPLE22, is substantially purified and administered according to theformulation of EXAMPLE 17 to a human patient afflicted with Lewis LungCarcinoma at between approximately 10 mg per day to approximately 100 mgper day, and in a preferred amount of 50 mg per day, until the growth ofthe tumor is inhibited.

EXAMPLE 26

[0253] Use of a Combinatorial Chemistry Screening Library

[0254] Synthetic studies of PLH are conducted to obtain more potent andless toxic agents. According to the method described by Gordon andSteele, J. Bioorg. Med. Chem. Letters (1995), 5, 47-50, phenylahistinare its derivatives are screened using an efficient solid phasecombinatorial synthesis for a typical diketopiperazine library. By usingsuch technique, phenylahistin is utilized as a prototype for thedevelopment of promising new antitumor agents.

EXAMPLE 27 Pharmaceutical Formulations of the Synthesized Phenylahistins

[0255] 1) Formulations Administered Intravenously by Drip, Injection,Infusion or the Like

[0256] Vials containing 5 g of powdered glucose are each addedaseptically with 10 mg of a compound synthesized by the method andsealed. After being charged with nitrogen, helium or other inert gas,the vials are stored in a cool, dark place. Before use, the contents aredissolved in ethanol and added to 100 ml of a 0.85% physiological saltwater solution. The resultant solution is administered as a method ofinhibiting the growth of a cancerous tumor in a human diagnosed ashaving such a tumor at between approximately 10 ml/day to approximately1000 ml/day, intravenously, by drip, or via a subcutaneous orintraperitoneal injection, as deemed appropriate by those of ordinaryskill in the art.

[0257] 2) Formulation to be Administered Orally or the Like

[0258] A mixture obtained by thoroughly blending 1 g of a compoundsynthesized by the method, 98 g of lactose and 1 g of hydroxypropylcellulose is formed into granules by any conventional method. Thegranules are thoroughly dried and sifted to obtain a granule preparationsuitable for packaging in bottles or by heat sealing. The resultantgranule preparations are orally administered at between approximately100 ml/day to approximately 1000 ml/day, depending on the symptoms, asdeemed appropriate by those of ordinary skill in the art of treatingcancerous tumors in humans.

[0259] 3) Formulation to be Administered Topically

[0260] Administration to an individual of an effective amount of thecompound can also be accomplished topically by administering thecompound(s) directly to the affected area of the skin of the individual.For this purpose, the compound administered or applied is in the form ofa composition including a pharmacologically acceptable topical carrier,such as a gel, an ointment, a lotion, or a cream, which includes,without limitation, such carriers as water, glycerol, alcohol, propyleneglycol, fatty alcohols, triglycerides, fatty acid esters, or mineraloils. Other topical carriers include liquid petroleum, isopropylpalmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurate (5%) in water, or sodium lauryl sulfate (5%) in water. Othermaterials such as anti-oxidants, humectants, viscosity stabilizers, andsimilar agents may be added as necessary. Percutaneous penetrationenhancers such as Azone may also be included. In addition, in certaininstances, it is expected that the compound may be disposed withindevices placed upon, in, or under the skin. Such devices includepatches, implants, and injections which release the compound into theskin, by either passive or active release mechanisms.

EXAMPLE 28 In Vitro Pharmacology of t-butyl Phenylahistin

[0261] The in vitro efficacy studies performed with t-butylphenylahistin included: A) a panel of six tumor cell lines, B) studiesin multidrug-resistant tumor cells, and C) studies to determine themechanism of action.

[0262] A). Study of t-butyl Phenylahistin in a Panel of Six Tumor CellLines

[0263] The following cell lines (source in parentheses) were used: HT29(human colon tumor; ATCC; HTB-38), PC3 (human prostate tumor; ATCC;CRL-1435), MDA-MB-231 (human breast tumor; ATCC; HTB-26), NCI-H292(human non-small cell lung tumor; ATCC; CRL-1848), OVCAR-3 (humanovarian tumor; ATCC; HTB-161), B16-F10 (murine melanoma; ATCC; CRL-6475)and CCD-27 sk (normal human fibroblast; ATCC; CRL-1475). Cells weremaintained at subconfluent densities in their respective culture media.

[0264] Cytotoxicity assays were performed as described above in Example4, using Resazurin fluorescence as an indicator of cell viability.

[0265] The disclosed compounds are effective agents against a variety ofdifferent and distinct tumor cell lines. Specifically, for example,t-butyl-phenylahistin exhibited its greatest potency against the PC-3tumor cell line, although the greatest efficacy was displayed againstthe HT-29 cell line. t-butyl-phenylahistin displayed a markeddifferential between normal fibroblasts and the tumor cell lines, with aratio ranging from >20->100, except for the OVCAR-3 cell line. TABLE 10Activity of t-butyl phenylahistin in the Tumor Panel Screent-butyl-phenylahistin Cell Line Mean SD n HT-29 Colon IC50 nM 420 473 3% Cytotoxicity 88 0.2 3 PC-3 Prostate IC50 nM 174 — 2 % Cytotoxicity59.5 — 2 MDA-MB-231 Breast IC50 nM 387 — 2 % Cytotoxicity 65.5 — 2NCI-H292 Lung IC50 nM 384 194 3 % Cytotoxicity 65 5 3 OVCAR-3 Ovary IC50nM >20,000 — 2 % Cytotoxicity 37 — 2 B16-F10 Melanoma IC50 nM 736 650 3% Cytotoxicity 74 2 3 CCD-27sk Fibroblast IC50 nM >20,000 — 2 %Cytotoxicity 45 — 2

[0266] B). Studies in Drug Resistant Cell Lines

[0267] One of the major challenges in the use of chemotherapeutic agentsin clinical oncology is the development of resistance to the drug effectby the tumor cells. There are several mechanisms for the development ofresistance, each of which will have differential effects onchemotherapeutic drugs. These mechanisms include increased expression ofATP-dependent efflux pumps such as the P-glycoprotein encoded by MDR1 orthe multidrug-resistance associated protein 1 encoded by MRP1. Reduceddrug uptake, alteration of the drug's target, increasing repair ofdrug-induced DNA damage, alteration of the apoptotic pathway and theactivation of cytochrome P450 enzymes are other examples of mechanismsby which cancer cells become resistant to anticancer drugs. The selectedcompounds were studied in three different cell lines that exhibit twodifferent mechanisms of resistance; the overexpression of theP-glycoprotein and altered topoisomerase II activity.

[0268] 1) Human Uterine Sarcoma Tumor Cell Line Pair: MES-SA (TaxolSensitive) and MES-SA DX (Taxol Resistant).

[0269] This cell line expresses elevated mdr-1 mRNA and P-glycoprotein(an extrusion pump mechanism). Pretreatment with cyclosporin-A (CsA)blocks P-glycoprotein and reinstates activity in the resistant cell linefor those compounds for which the resistance is due to elevatedP-glycoprotein.

[0270] As can be seen from Table 11, the potency oft-butyl-phenylahistin was only slightly reduced. Cyclosporin A (CsA)pretreatment did not alter the potency of the selected compounds. Incontrast, taxol was virtually inactive in the MES-SA DX resistant cellline, whereas this compound was very potent in the sensitive cell line.CsA treatment restored the sensitivity to taxol of the MES-SA DX cellline. The MES-SA DX cell line also showed reduced susceptibility toetoposide (60 fold), doxorubicin (34 fold) and mitoxantrone (20 fold).

[0271] These data indicate that the effects of t-butyl-phenylahistin arenot susceptible to the taxol-related resistance mechanism(p-glycoprotein) in this cell line, and that cross-resistance from taxoldoes not occur to this compound in this model. TABLE 11 Activity oft-butyl-phenylahistin and Taxol in MES-SA Taxol Sensitive and MES-SA DXTaxol Resistant Human Uterine Sarcoma Tumor Cell Lines MES-SA SensitiveNo MES-SA DX Resistant Com- CsA CsA Pretreat No CsA CsA Pretreat poundIC50 IC50 Ratio IC50 Ratio IC50 Ratio Study nM nM No CsA nM MES-SA nM NoCsA t-butyl- phenyl- ahistin Study I 144 — — 825 5.7 — — Study III 122162 1.3 694 4.3 622 0.9 Taxol Study I 4.4 — — >20,000 >455 — — Study II13.3 7.6 0.6 >>100 >>8 40 <<0.25 Study III 7.3 2.8 0.4 >24,000 >3000 2.0<<0.001

[0272] 2) Human Acute Promyelocytic Leukemia Cell Line Pair: HL-60(Mitoxantrone-Sensitive) and HL-60/MX-2 (Mitoxantrone-Resistant)

[0273] This cell line is considered to have atypical drug resistanceproperties with altered topoisomerase II catalytic activity withoutoverexpression of P-glycoprotein.

[0274] As can be seen in Table 12, these results indicate that thepotency of the novel compound in the sensitive and resistant HL-60 celllines. In contrast, Mitoxantrone loses efficacy by a factor of 24-foldin the resistant HL-60/MX-2 cell line.

[0275] Thus, t-butyl-phenylahistin is not susceptible to the sameresistance mechanisms as Mitoxantrone in this cell line, and there is nocross-resistance from Mitoxantrone to t-butyl phenylahistin in thismodel. TABLE 12 Activity of t-butyl-phenylahistin and Mitoxantrone inthe HL-60 Human Acute Promyelocytic Leukemia Tumor Sensitive andResistant Cell Line Pair HL-60 HL-60 Resistant Sensitive Ratio CompoundIC50 nM IC50 nM to Sensitive t-butyl-phenylahistin 255 175 0.69Mitoxantrone 202 4870 24.1

[0276] C) Studies of the Mechanism of Action

[0277] 1). Action on Microtubule Function

[0278] Human umbilical vein endothelial cells (HuVEC from Cambrex) wereused in this study, for evaluating the effects of t-butyl-phenylahistinin comparison to colchicine and taxol on tubulin by staining forα-tubulin.

[0279] Thirty minutes exposure to t-butyl-phenylahistin or colchicine(all at 2 μM) induced microtubule depolymerization as was indicated bythe lack of intact microtubule structure in contrast to that observed inthe DMSO Control and cell membrane blebbing (a clear indication ofapoptosis) in the HuVEC cells, whereas taxol did not induce microtubuledepolymerization under these conditions. Colchicine is a knownmicrotubule depolymerizing agent whereas taxol is a tubulin stabilizingagent.

[0280] 2). Activation of the Caspase Cascade

[0281] Several enzymes in the caspase cascade are activated duringapoptosis, including Caspase-3, -8 and-9. The activity of Caspase-3 wasmonitored in Jurkat cells following treatment witht-butyl-phenylahistin.

[0282] The results indicate that caspase-3 was activated in adose-dependent manner by treatment with t-butyl-phenylahistin in amanner similar to halimide. The caspase-3 activation occurred over asimilar concentration range as for the IC50s for cytotoxicity in theJurkat cell line (Table 13). TABLE 13 Cytotoxicity oft-butyl-phenylahistin in Jurkat Cells Cytotoxicity Potency Efficacy NPICompound IC50 nM % Cell Death t-butyl-phenylahistin 165 93 Mitoxantrone41 99

[0283] 3). Cleavage of Poly(ADP-Ribose) Polymerase (PARP) in JurkatCells

[0284] In order to assess the ability of t-butyl-phenylahistin to induceapoptosis in Jurkat cells, cleavage of poly(ADP-ribose) polymerase(PARP) was monitored. PARP is a 116 kDa nuclear protein that is one ofthe main intracellular targets of Caspase-3. The cleavage of PARPgenerates a stable 89 kDa product, and this process can be easilymonitored by western blotting. Cleavage of PARP by caspases is one ofthe hallmarks of apoptosis, and as such serves as an excellent markerfor this process. Halimide was active at inducing cleavage of PARP inJurkat cells 10 hours after exposure of the cells to the compound.

[0285] 4). Enhanced Vascular Permeability in HuVEC Cells

[0286] Compounds that depolymerize microtubules (e.g. combretastatinA-4-phosphate, ZD6126) have been shown to induce vascular collapse intumors in vivo. This vascular collapse is preceded by a rapid inductionof vascular cell permeability initially to electrolytes and soon afterto large molecules. The enhanced permeability of HuVEC cells to afluorescent-labeled dextran is used as a proxy assay for vascularcollapse.

[0287] t-butyl-phenylahistin rapidly (within 1 hour) induced significantHuVEC monolayer permeability, to an extent similar to colchicine. Themicrotubule stabilizing agent taxol was inactive in this assay (FIG.12).

EXAMPLE 29 In Vivo Pharmacology

[0288] Studies with t-butyl-phenylahistin were performed. The novelcompound was studied as monotherapy and in combination with aclinically-used chemotherapeutic agent. The doses of the selected novelcompound were determined from the acute tolerability testing (MaximallyTolerated Dose, MTD) and were adjusted if necessary during each study.The doses of the clinically-used chemotherapeutic agents were selectedon the basis of historical studies.

[0289] t-butyl-phenylahistin was were compared in the HT-29 human colontumor, the DU-145 human prostate and the MCF-7 human breast tumorxenograft models.

[0290] The above models all use the subcutaneous xenograft implantationtechnique and are potentially subject to selective effects of a compoundon the subcutaneous vasculature producing a magnified (or apparent)antitumor activity. In order to circumvent this possibility, two othertumor models have been incorporated in the research. One of these is theobservation of lung metastases following the intravenous injection ofB16-F10 mouse melanoma tumor cells. The other model is the implantationof MDA-231 human breast tumor cells in the mouse mammary fat pad. Whilethis latter model is a xenograft model, the subcutaneous vasculaturedoes not play a role.

[0291] Methods

[0292] 1). Xenograft Models

[0293] Animals used were (exceptions are indicated for individualstudies): female nude mice (nu/nu) between 5 and 6 weeks of age (˜20 g,Harlan); group size was 9-10 mice per group unless otherwise indicated.

[0294] Cell lines used for tumor implantation were: HT-29 human colontumor; MCF-7 human breast tumor; A549 human non small cell lung tumor;MiaPaCa-2 human pancreas tumor; DU-145 human prostate tumor.

[0295] The selected novel compound was administered as monotherapy viathe intraperitoneal (i.p.) route at the doses indicated for theindividual study; for the combination studies the selected referencechemotherapy agents were injected 15-30 min prior to the compound.

[0296] Vehicles used in these studies were: 12.5% DMSO, 5% Cremaphor and82.5% peanut oil for the selected novel compounds; (1:3) Polysorbate80:13% ethanol for taxotere; (1:1) Cremaphor:ethanol for paclitaxel; forCPT-11 each mL of solution contained 20 mg of irinotecan hydrochloride,45 mg of sorbitol NF powder, and 0.9 mg of lactic acid, the pH beingadjusted to 7.4 with NaOH or HCl. Saline dilutions are used to achievethe injection concentrations used for the reference compounds.

[0297] HT-29 Human Colon Tumor Model

[0298] Animals were implanted subcutaneously (s.c.) by trocar withfragments of HT-29 tumors harvested from s.c. growing tumors in nudemice hosts. When the tumor size reached 5 mm×5 mm (about 10-17 days) theanimals were matched into treatment and control groups. Mice wereweighed twice weekly and tumor measurements were obtained using caliperstwice weekly, starting on Day 1. The tumor measurements were convertedto estimated mg tumor weight using the formula (W²×L)/2. When theestimated tumor weight of the control group reached an average of 1000mg the mice were weighed, sacrificed and the tumor removed. The tumorswere weighed and the mean tumor weight per group was calculated and thetumor growth inhibition (TGI) was determined for each group (100% minusthe change in the mean treated tumor weight/the change in the meancontrol tumor weight×100.

[0299] In this model unless otherwise noted for the individual study,the selected novel compound was injected intraperitoneally every thirdday for 15 days [1, 4, 8, 11 and 15 (q3d×5)]; CPT-11 was administeredintraperitoneally on days 1, 8 and 15 (qw×3).

[0300] MCF-7 Human Breast Tumor Model

[0301] Female nude mice (˜20 g) were implanted s.c. with 21-day releaseestrogen (0.25 mg) pellets 24 hours prior to the s.c. implantation withMCF-7 tumor fragments (harvested from s.c. tumors in nude mice hosts).The study then proceeded as described for the HT-29 model, usingtaxotere as the standard chemotherapy agent.

[0302] In this model unless otherwise noted for the individual study,the novel compound was injected via the intraperitoneal route daily onDays 1-5, inclusive (qd×5); taxotere was administered intravenously onDays 1, 3 and 5 (qod×3).

[0303] A549 Human Lung Tumor Model

[0304] Animals were implanted s.c. by trocar with fragments of A549tumors harvested from s.c. growing tumors in nude mice hosts. When thetumor size reached 5 mm×5 mm (about 10-17 days) the animals were matchedinto treatment and control groups. The rest of the study proceeded asdescribed for the HT-29 model, using taxotere and CPT-11 as the standardchemotherapy agents.

[0305] In this model unless otherwise noted for the individual study,the tested compound was administered via the intraperitoneal route on aq3d×5 dose schedule for the CPT-11 combination or on a qd×5 dose regimenfor the combination with taxotere; CPT-11 was administered via theintraperitoneal route on a qw×3 schedule; taxotere was administeredintravenously on a qod×3 dose regimen.

[0306] MiaPaCa-2 Human Pancreas Tumor Model

[0307] Animals were implanted s.c. by trocar with fragments of MiaPaCa-2tumors harvested from s.c. growing tumors in nude mice hosts. When thetumor size reached 5 mm×5 mm (about 10-17 days) the animals were matchedinto treatment and control groups. The rest of the study proceeded asdescribed for the HT-29 model, using gemcitabine as the standardchemotherapy agent.

[0308] In this model unless otherwise noted for the individual study,test compound was administered every third day via the intraperitonealroute on Days 1, 4, 7, 10 and 15 (q3d×5); gemcitabine was administeredvia the intraperitoneal route on Days 1, 4, 7 and 10 (q3d×4).

[0309] DU-145 Human Prostate Tumor Model

[0310] Male mice were implanted s.c. by trocar with fragments of DU-145tumors harvested from s.c. growing tumors in nude male mice hosts. Whenthe tumors reached ˜5 mm×5 mm ( at about 13-17 days) the animals werematched into treatment and control groups. The remainder of the studyproceeded as for the HT-29 model, using taxotere as the standardchemotherapy agent.

[0311] In this model unless otherwise noted for the individual study,test compound was administered via the intraperitoneal route on Days 1,3, 5, 8 and 11 (q3d×5); taxotere was administered intravenously on Days1, 3 and 5 (q2d×3).

[0312] 2). Non Subcutaneous Implantation Tumor Models

[0313] The animals used were: female nude mice (nu/nu) (MDA-231 study)or B6D2F1 (B16-F10 studies) mice between 5 and 6 weeks of age (˜20 g,Harlan); group size was 10 mice per group unless otherwise indicated.

[0314] The cell lines used were: MDA-MB-231 human breast tumor andB16-F10 murine melanoma cells.

[0315] Compound was administered as monotherapy via the intraperitonealroute at the doses indicated for the individual study; for thecombination studies the selected reference chemotherapy agents wereinjected 15-30 min prior to the NPI compound.

[0316] MDA-231 Human Breast Tumor

[0317] Female nude mice were injected in the mammary fat pad with 2×10⁶MDA-231 cells harvested from in vitro cell culture. When the tumor sizereached 5 mm×5 mm (about 14-28 days) the animals were matched intotreatment and control groups. The study then proceeded as described forthe HT-29 model, using paclitaxel as the standard chemotherapy agent.

[0318] In this model unless otherwise noted for the individual study,the test compound was administered via the intraperitoneal route on Days1, 4, 8, 11 and 15 (q3d×5); paclitaxel was administered via theintraperitoneal route on Days 1-5 (qd×5).

[0319] B16-F10 Metastatic Murine Melanoma Model

[0320] Mice received B16-F10 cells (prepared from an in vitro cellculture of B16-F10 cells) by the iv route on Day 0. On Day 1 mice wererandomized into treatment and control groups and treatment commenced.Mice were weighed twice weekly, starting on Day 1. All mice aresacrificed on Day 16, the lungs removed, weighed and the surfacecolonies counted. Results are expressed as mean colonies of treatedmice/mean colonies of control mice (T/C)×100%). The metastasis growthinhibition (MGI) is this number subtracted from 100%. Paclitaxel was thestandard chemotherapy agent used in this study.

[0321] In this model unless otherwise noted for the individual study,the test compounds were administered via the intraperitoneal route onDays 1-5 (qd×5); paclitaxel was administered intravenously on Days1-5(qd×5).

[0322] When appropriate (n 3), results are presented as means ± SEM.Statistical analysis of studies with several groups was performed usingANOVA with Neuman-Keuls post test, unless otherwise indicated. Aone-tailed t-test was also used based on the hypothesis that thecompound or drug, or the combination, would reduce tumor growth.

Results Studies in the HT-29 Human Colon Tumor Xenograft Model

[0323] 1. Study of Activity of t-butyl-Phenyalhistin in the HT-29 HumanColon Tumor Xenograft Study

[0324] The results of this study are presented in FIG. 17 and Table 14.The combination therapy group indicated a marked synergy between thenovel compound and CPT-11. The individual tumor weights demonstrate theeffectiveness of the cotherapy treatment (FIG. 18). The TGI for thecombination group surpasses the NCI criterion for a positive effect,whereas the TGI for CPT-11 monotherapy did not reach this level. TABLE14 Summary of Studies Performed in the HT-29 Human Colon Tumor ModelChemotherapeutic Combination Study Description NPI-Compound Agent ExceedNCI Number Number, Result Name, Result Results Criterion Status Endpointmg/kg ip TGI % Dose TGI % TGI % (TGI 58%) Comments 2139 TGI t-butyl- NoEffect CPT-11 32.7 77.7 Combination Synergy phenylahistin 100 ip *, # 30qwx3 q3dx5

[0325] 2. Summary of the Effects of t-butyl-Phenylahistin in Combinationwith CPT-11 in the HT-29 Human Colon Tumor Xenograft Model

[0326] When combined with CPT-11, t-butyl-phenylahistin enhanced theeffect of CPT-11, the standard chemotherapeutic agent, to a level wellin excess of the NCI criterion of a TGI 58% for a positive effect. Theresults generated in the study are very comparable for both the in-lifeobservations and for the weights of the tumors excised at autopsy.

Studies in the DU-145 Human Prostate Tumor Xenograft Model

[0327] The study involved an assessment of t-butyl-phenyalhistin aloneand in combination with taxotere.

[0328] 1. Activity of t-butyl-phenylahistin Alone or in Combination withTaxotere in the DU-145 Human Prostate Xenograft Model

[0329] A study comparing t-butyl-phenylahistin alone and in combinationwith taxotere was initiated. The results are shown in FIG. 9.

[0330] The excised tumor weights at autopsy confirmed the observationsmade during the in-life segment of the study. The tumor growthinhibition indices indicated a marked inhibition of tumor growth.Taxotere alone did not reach the NCI established criterion for apositive effect (TGA 58%).

[0331] 2. Studies in the MCF-7 Human Breast Tumor Xenograft Model

[0332] This study considered the effects of t-butyl-phenylahistin in theMCF-7 human breast tumor xenograft model. The doses of the compound wereadministered on Days 1, 2, 3, 4, and 7; Taxotere was administered onDays 1, 3 and 7.

[0333] The selected novel compound has early onset, statisticallysignificant effects when used in combination with taxotere in thismodel, apparently almost completely blocking estimated tumor growth(FIG. 10). t-butyl-phenylahistin exhibited a significant potentiation oftaxotere.

[0334] 3. Studies in the Murine Melanoma B16 F10 Metastatic Tumor Model

[0335] This study examined the effect of t-butyl-phenylahistin alone andin combination with paclitaxel on the number of metastases appearing onthe surface of the lung 16 days after the intravenous injection of B16F10 melanoma cells to the mouse. This model is not a xenograft model;however, it does not involve a high degree of vascularization into thetumor mass.

[0336] This study does not itself establish that combination therapy ismore effective than monotherapy, it does indicate thatt-butyl-phenylahistin is most effective in highly vascularized tumors(FIG. 12).

EXAMPLE 30 Assays for Activity Against Pathogenic Fungi

[0337] Comparative activity of a phenylahistin or its analog against apathogenic fungus, relative to known antifungal compounds recited above,for use in determining the phenylahistin or its analog's AF/IS value ismeasured directly against the fungal organism, e.g. by microtiter plateadaptation of the NCCLS broth macrodilution method described in DiagnMicro and Infect Diseases 21:129-133 (1995). Antifungal activity canalso be determined in whole-animal models of fungal infection. Forinstance, one may employ the steroid-treated mouse model of pulmonarymucormycosis (Goldaill, L. Z. & Sugar, A. M. 1994 J Antimicrob Chemother33:369-372). By way of illustration, in such studies, a number ofanimals are given no phenylahistin or its analog, various doses ofphenylahistin or its analog (and/or combinations with one or more otherantifungal agents), or a positive control (e.g. Amphotericin B),respectively, beginning before, at the time of, or subsequent toinfection with the fungus. Animals may be treated once every 24 hourswith the selected dose of phenylahistin or its analog, positive control,or vehicle only. Treatment is continued for a predetermined number ofdays, e.g. up to ten days. Animals are observed for some time after thetreatment period, e.g. for a total of three weeks, with mortality beingassessed daily. Models can involve systemic, pulmonary, vaginal andother models of infection with or without other treatments (e.g.treatment with steroids) designed to mimic a human subject susceptibleto infection.

[0338] To further illustrate, one method for determining the in vivotherapeutic efficacies (ED₅₀, e.g. expressed in mg phenylahistin or itsanalog/kg subject), is a rodent model system. For example, a mouse isinfected with the fungal pathogen such as by intravenous infection withapproximately 10 times the 50% lethal dose of the pathogen (10⁶ C.albicans cells /mouse). Immediately after the fungal infection,phenylahistin compounds are given to the mouse at a predetermined dosedvolume. The ED₅₀ is calculated by the method of Van der Waerden (ArchExp Pathol Pharmakol 195:389-412, 1940) from the survival rate recordedon 20th day post-infection. Generally, untreated control animals die 7to 13 days post-infection.

[0339] In another illustrative embodiment, C. albicans Wisconsin (C43)and C. tropicalis (C112), grown on Sabouraud dextrose agar (SDA) slantsfor 48 h at 28° C., are suspended in saline and adjusted to 46%transmission at 550 nm on a spectrophotometer. The inoculum is furtheradjusted by hemacytometer and confirmed by plate counts to beapproximately 1 or 5×10⁷ CFU/ml. CF-1 mice are infected by injection 1or 5×10⁶ CFU into the tail vein. Antifungal agents are administeredintravenously or subcutaneously in ethanol:water (10:90), 4 h postinfection and once daily thereafter for 3 or 4 more days. Survival ismonitored daily. The ED₅₀ can be defined as that dose which allows for50% survival of mice.

EXAMPLE 31 Evaluating Antimicotic Activity

[0340] Benzimidazoles and griseofulvin are anti-tubulin agents capableof binding to fungal microtubules. Once bound, these compounds interferewith cell division and intracellular transport in sensitive organisms,resulting in cell death. Commercially, benzimidazoles are used asfungicidal agents in veterinary medicine and plant disease control. Awide variety of fungal species, including Botrytis cinerea, Beauveriabassiana, Helminthosporium solani, Saccharomyces cerevisiae andAspergillus are susceptible to these molecules. Toxicity concerns andincreasing drug resistance, however, have negatively impacted theirusage. Griseofulvin is used clinically to treat ringworm infections ofthe skin, hair and nails, caused by Trichophyton sp., Microsporum sp.,and Epidermophyton floccosum. Its antifungal spectrum, however, isrestricted to this class of fungal organisms. Genotoxicity is also asignificant side effect. Terbinafine, while an alternative first-linetreatment, is more costly. Further, clinical resistance recently hasbeen observed in Trichophyton rubrum (the major causative agent for alldermatophyte infections).

[0341] In Candida albicans, microtubule/microfilament formation isaffected where cells are exposed to the microtubule inhibitorsnocodazole and chloropropham. These results further validate theexploration of cytoskeleton inhibitors as effective antimycotic agents.Accordingly, several of the compounds disclosed herein were evaluatedfor antimycotic activity.

[0342] Specifically, disclosed compounds were evaluated alongsidecommercially available microtubulin inhibitors as well as recognizedantifungal agents. The test compounds and controls used in this study:(−)-Phenylahistin and t-butyl phenylahistin, Colchicine (commercialmicrotubulin inhibitor tested versus 3 Candida isolates), Benomyl(commercial microtubulin inhibitor tested versus 3 Candida isolates),Griseofulvin (commercial microtubulin inhibitor and antibiotic controlfor testing versus 6 dermatophyte isolates), Amphotericin B (antibioticcontrol for testing versus 3 Candida isolates), Itraconazole (antibioticcontrol for testing versus 2 Aspergillus isolates).

[0343] Microorganisms against which these compounds were testedincluded: Candida albicans, Candida glabrata, Aspergillus fumigatus,Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophytonfloccosum. With the exception of Candida glabrata (one isolate), twoisolates of each species were tested.

[0344] Antifungal susceptibility testing was accomplished according tothe methods outlined in the National Committee for Clinical LaboratoryStandards, M38-A “Reference Method for Broth Dilution AntifungalSusceptibility Testing of Conidium-Forming Filamentous Fungi; ApprovedStandard.” This includes testing in RPMI-1640 with glutamine and withoutbicarbonate, an inoculum size of 0.4-5×10⁴, and incubation at 30 or 35°C. for 48 hours. The minimum inhibitory concentration (MIC) was definedas the lowest concentration that resulted in an 80% reduction inturbidity as compared to a drug-free control tube. Drug concentrationswere 0.03-16 μg/ml for the investigational compounds, 0.015-8 μg/ml foritraconazole and griseofulvin.

[0345] The minimum inhibitory concentration (MIC) at which a compoundprevented the growth of the target microorganism was assessed accordingto the modified version of the NCCLS protocol. Minimum inhibitoryconcentrations (MIC) were determined at the first 24-hour interval wheregrowth could be determined in the drug-free control tube. The definedMIC was the lowest concentration that exhibited an 80% reduction inturbidity as compared to the growth control. The minimum lethalconcentration (MLC) was determined by plating 0.1 μl from the MICconcentration and each concentration above the MIC. The MLC was calledat the first concentration that exhibited five or fewer colonies offungal growth representing a 99.95% kill. When a MIC was obtained, aminimum fungicidal concentration (MFC) was determined to assess thefungistatic/fungicidal nature of the compound. This procedure entailsdiluting drug-treated cell samples (removed from test wells containingcompound at and above the MIC) to compound concentrations significantlybelow the inhibitory concentration and depositing them on agar plates.The compound is scored as fungistatic if the cells are able to resumegrowth and fungicidal if no regrowth is possible because the compoundhad killed the organisms.

[0346] Compounds disclosed herein were shown to be effective against twoTrichophyton species. T. rubrum is the principal causative agent forhuman dermatophytic infections, and would be the key organism to targetin the development of a clinical agent.

[0347] Compound t-butylphenylahistin at least was equivalent in potencycompared to griseofulvin, a current, standard pharmaceutical agent usedfor treating dermatophytic infections.

[0348] Compound (−)-Phenylahistin also was potent when tested versus T.rubrum and even more potent versus the sensitive T. mentagrophytesisolate.

[0349] In those instances when an MFC could be determined, the resultsindicate that these compounds are fungistatic in nature (see Tables 19and 20). TABLE 17 Antifungal Activity of Phenylahistins MICs and MFCs,μg/ml C. albicans C. albicans C. A. fumigatus A. fumigatus 90028 10231glabrata isolate #1 isolate #2 Compound MIC MFC MIC MFC MIC MFC MIC MFCMIC MFC (−)-Phenylahistin >70  ND** >70* ND >70 ND >16 ND >16 ND t-butylphenylahistin >32 ND >32 ND >32 ND >16 ND <0.03 0.125 amphotericin B 0.50.5 0.5 0.5 1 1 ND ND ND ND griseofulvin ND ND ND ND ND ND ND ND 0.5 NDitraconazole ND ND ND ND ND ND 1 ND ND ND colchicine >128 ND >128ND >128 ND ND ND ND ND benomyl 64 >512 64 >512 64 >512 ND ND ND ND

[0350] TABLE 18 Antifungal Activity of Phenylahistins MICs and MFCs,μg/ml T. menta- T. menta T. rubrum T. rubrum grophytes grophytes E.floccosum E. floccosum isolate #1 isolate #2 isolate #1 isolate #2isolate #1 isolate #2 Compound MIC MFC MIC MFC MIC MFC MIC MFC MIC MFCMFC NPI2350 >16 ND 0.16 >16 16 >16 >16 ND >16 ND >16 ND NPI2460 <0.030.125 <0.03 <0.03 4 >16 >16 ND >16 ND >16 ND amphotericin B ND ND ND NDND ND ND ND ND ND ND ND griseofulvin 0.5 ND <0.015 ND 1 ND 2 ND 2 ND 4ND itraconazole ND ND ND ND ND ND ND ND ND ND ND ND colchicine ND ND NDND ND ND ND ND ND ND ND ND benomyl ND ND ND ND ND ND ND ND ND ND ND ND

[0351] The examples described above are set forth solely to assist inthe understanding of the invention. Thus, those skilled in the art willappreciate that the disclosed methods and compounds encompass and mayotherwise provide further derivatives of phenylahistins.

[0352] One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain, for example, the ends andadvantages mentioned, as well as others inherent. The methods andprocedures described herein are presently representative of preferredembodiments and are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention.

[0353] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention.

[0354] As noted above, all patents and publications mentioned in thespecification are indicative of the levels of those skilled in the artto which the invention pertains. All patents and publications are herebyincorporated by reference herein to the extent allowable by law, suchthat each individual patent and publication may be treated asspecifically and individually indicated to be incorporated by reference.

[0355] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein. The terms andexpressions which have been employed are used as terms of descriptionand not of limitation, and there is no intention that in the use of suchterms and expressions indicates the exclusion of equivalents of thefeatures shown and described or portions thereof. It is recognized thatvarious modifications are possible within the scope of the invention.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be falling within thescope of the invention.

What is claimed is:
 1. A method of treating and/or preventing at leastone fungal infection in a mammal afflicted with at least one fungalinfection which comprises administering an antifungally effective amountof a compound sufficient for such treating or preventing, and whereinthe compound has the following structure:

wherein: R₁, R₂, R₅, R₇, and R₈ are each separately selected from thegroup consisting of a hydrogen atom, a halogen atom, and saturatedC₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, amino, substituted amino, nitro, substituted nitro, phenyl,and substituted phenyl groups, R₃, R₄, and R₆ are each separatelyselected from the group consisting of a hydrogen atom, a halogen atom,and saturated C₁-C₁₂ alkyl, unsaturated C₁-C₁₂ alkenyl, cycloalkyl,alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, amino, substituted amino, nitro, and substituted nitrogroups, X₁ and X₂ are separately selected from the group consisting ofan oxygen atom, and a sulfur atom, and the dashed bond represents a bondselected from the group consisting of a carbon-carbon single bond and acarbon-carbon double bond.
 2. The method of claim 1, wherein the fungalinfection is selected from the group consisting of an Aspergillosisinfection, blastomycosis infection, Candidiasis infection,Coccidioidomycosis infection, Cryptococcosis infection, Histopolasmosisinfection, Paracoccidioidomycosis, Sporotrichosisand, and Mucormycosisinfection.
 3. The method of claim 2, wherein the Aspergillosis infectionis invasive pulmonary aspergillosis.
 4. The method of claim 2, whereinthe Mucormycosis infection is craniofacial mucormycosis or pulmonarymucormycosis.
 5. The method of claim 2, wherein the Candidiasisinfection is retrograde candidiasis of the urinary tract.
 6. Apharmaceutical composition for treating or preventing fungal infectioncomprising an antifungally effective amount of a pharmaceuticallyacceptable carrier and a compound having the structure:

wherein: R₁, R₂, R₅, R₇, and R₈ are each separately selected from thegroup consisting of a hydrogen atom, a halogen atom, and saturatedC₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, amino, substituted amino, nitro, substituted nitro, phenyl,and substituted phenyl groups, R₃, R₄, and R₆ are each separatelyselected from the group consisting of a hydrogen atom, a halogen atom,and saturated C₁-C₁₂ alkyl, unsaturated C₁-C₁₂ alkenyl, cycloalkyl,alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, amino, substituted amino, nitro, and substituted nitrogroups, X₁ and X₂ are separately selected from the group consisting ofan oxygen atom, and a sulfur atom, and the dashed bond represents a bondselected from the group consisting of a carbon-carbon single bond and acarbon-carbon double bond.
 7. The composition of claim 6, wherein thefungal infection is selected from the group consisting of anAspergillosis infection, blastomycosis infection, Candidiasis infection,Coccidioidomycosis infection, Cryptococcosis infection, Histopolasmosisinfection, Paracoccidioidomycosis, Sporotrichosisand, and Mucormycosisinfection.
 8. The composition of claim 7, wherein the Aspergillosisinfection is invasive pulmonary aspergillosis.
 9. The composition ofclaim 7, wherein the Mucormycosis infection is craniofacial mucormycosisor pulmonary mucormycosis.
 10. The composition of claim 7, wherein theCandidiasis infection is retrograde candidiasis of the urinary tract.